lipofectamine 2000  (Thermo Fisher)


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    Name:
    Lipofectamine 2000 Transfection Reagent
    Description:
    Lipofectamine 2000 Transfection Reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines Researchers use Lipofectamine 2000 Reagent for siRNA and shRNA based gene knockdown experiments as well as for gene expression studies With Lipofectamine 2000 Transfection Reagent you ll get • Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression see table • Superior performance for co transfection of siRNA and plasmid DNA• Proven efficacy in the presence of serum eliminates the need to change media following transfection• Reliable performance for high throughput applications• The best choice for establishing stable cell linesA high performance transfection reagent for gene expression and gene silencingLipofectamine 2000 Transfection Reagent works effectively with all common cell lines as well as with many challenging ones and can be used in media with or without serum For gene silencing Lipofectamine 2000 Transfection Reagent s high efficiency transfections provide the high levels of gene knockdown needed to produce convincing results Lipofectamine 2000 Transfection Reagent is the number one choice for co transfection given its effectiveness for transfecting both siRNA and plasmid DNA Lipofectamine 2000 Transfection Reagent is easy to use simply mix with nucleic acid and add to cell culture Ideal for high throughput workSimplicity and speed combined with high transfection efficiency make Lipofectamine 2000 Transfection Reagent ideal for transient protein expression or high throughput RNAi transfections Transfection conditions can be easily established for automated or robotic systems used in such applications
    Catalog Number:
    11668019
    Price:
    None
    Applications:
    Cell Culture|Plasmid Transfection|RNAi Transfection|Stem Cell & Primary Cell Transfections|Synthetic siRNA Transfection|shRNA & miR RNAi Plasmid Transfection|Transfection
    Category:
    Cell Culture Transfection Reagents
    Buy from Supplier


    Structured Review

    Thermo Fisher lipofectamine 2000
    BBR-induced G0/G1 phase cell cycle arrest is attributed to FoxO3a up-regulation in Huh-7 cells. ( A ) Knockdown of FoxO3a induces down-regulation of p21 Cip1 and p27 Kip1 , up-regulation of Skp2. Huh-7 cells were transfected with control and FoxO3a siRNA with <t>lipofectamine</t> 2000 for 48 h, followed by exposure the cells to berberine (120 μM) for an additional 24 h. The protein levels of FoxO3a, p21 Cip1 , p27 Kip1 and Skp2 were examined by western blot. Bar graphs demonstrate relative expression levels of indicated proteins. ## represents p
    Lipofectamine 2000 Transfection Reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines Researchers use Lipofectamine 2000 Reagent for siRNA and shRNA based gene knockdown experiments as well as for gene expression studies With Lipofectamine 2000 Transfection Reagent you ll get • Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression see table • Superior performance for co transfection of siRNA and plasmid DNA• Proven efficacy in the presence of serum eliminates the need to change media following transfection• Reliable performance for high throughput applications• The best choice for establishing stable cell linesA high performance transfection reagent for gene expression and gene silencingLipofectamine 2000 Transfection Reagent works effectively with all common cell lines as well as with many challenging ones and can be used in media with or without serum For gene silencing Lipofectamine 2000 Transfection Reagent s high efficiency transfections provide the high levels of gene knockdown needed to produce convincing results Lipofectamine 2000 Transfection Reagent is the number one choice for co transfection given its effectiveness for transfecting both siRNA and plasmid DNA Lipofectamine 2000 Transfection Reagent is easy to use simply mix with nucleic acid and add to cell culture Ideal for high throughput workSimplicity and speed combined with high transfection efficiency make Lipofectamine 2000 Transfection Reagent ideal for transient protein expression or high throughput RNAi transfections Transfection conditions can be easily established for automated or robotic systems used in such applications
    https://www.bioz.com/result/lipofectamine 2000/product/Thermo Fisher
    Average 99 stars, based on 75628 article reviews
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    Images

    1) Product Images from "Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells"

    Article Title: Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19020327

    BBR-induced G0/G1 phase cell cycle arrest is attributed to FoxO3a up-regulation in Huh-7 cells. ( A ) Knockdown of FoxO3a induces down-regulation of p21 Cip1 and p27 Kip1 , up-regulation of Skp2. Huh-7 cells were transfected with control and FoxO3a siRNA with lipofectamine 2000 for 48 h, followed by exposure the cells to berberine (120 μM) for an additional 24 h. The protein levels of FoxO3a, p21 Cip1 , p27 Kip1 and Skp2 were examined by western blot. Bar graphs demonstrate relative expression levels of indicated proteins. ## represents p
    Figure Legend Snippet: BBR-induced G0/G1 phase cell cycle arrest is attributed to FoxO3a up-regulation in Huh-7 cells. ( A ) Knockdown of FoxO3a induces down-regulation of p21 Cip1 and p27 Kip1 , up-regulation of Skp2. Huh-7 cells were transfected with control and FoxO3a siRNA with lipofectamine 2000 for 48 h, followed by exposure the cells to berberine (120 μM) for an additional 24 h. The protein levels of FoxO3a, p21 Cip1 , p27 Kip1 and Skp2 were examined by western blot. Bar graphs demonstrate relative expression levels of indicated proteins. ## represents p

    Techniques Used: Transfection, Western Blot, Expressing

    2) Product Images from "Functionalized Asymmetric Bola-Type Amphiphiles for Efficient Gene and Drug Delivery"

    Article Title: Functionalized Asymmetric Bola-Type Amphiphiles for Efficient Gene and Drug Delivery

    Journal: Nanomaterials

    doi: 10.3390/nano8020115

    The uptake cell percentage (columns, Cy5-positive cells) and mean fluorescence intensity (dots and lines) of bolaplexes at the optimal N/P ratio in HEK 293 ( A ) and HeLa ( B ) cells quantified by flow cytometry. Lipofectamine 2000 was used as the control. Data represent mean ± SD ( n = 3).
    Figure Legend Snippet: The uptake cell percentage (columns, Cy5-positive cells) and mean fluorescence intensity (dots and lines) of bolaplexes at the optimal N/P ratio in HEK 293 ( A ) and HeLa ( B ) cells quantified by flow cytometry. Lipofectamine 2000 was used as the control. Data represent mean ± SD ( n = 3).

    Techniques Used: Fluorescence, Flow Cytometry, Cytometry

    Fluorescent microscopy images of HEK 293 cells transfected by bolaplexes at the optimal N/P ratio (with the vector name and N/P ratio in each image). The molar ratio of bolaamphiphile/DOPE was 1:1, lipofectamine 2000 was used for comparison. The cells were observed by fluorescence microscopy after a 24 h transfection. Scale bar: 100 μm.
    Figure Legend Snippet: Fluorescent microscopy images of HEK 293 cells transfected by bolaplexes at the optimal N/P ratio (with the vector name and N/P ratio in each image). The molar ratio of bolaamphiphile/DOPE was 1:1, lipofectamine 2000 was used for comparison. The cells were observed by fluorescence microscopy after a 24 h transfection. Scale bar: 100 μm.

    Techniques Used: Microscopy, Transfection, Plasmid Preparation, Fluorescence

    3) Product Images from "Nanoscale polysaccharide derivative as an AEG-1 siRNA carrier for effective osteosarcoma therapy"

    Article Title: Nanoscale polysaccharide derivative as an AEG-1 siRNA carrier for effective osteosarcoma therapy

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S147747

    Electrophoretic migration of AEG-1 siRNA, Cs- g -PLLD/siAEG-1 ( A ), and Cs- g -PLLD-FA/siAEG-1 ( B ) complexes at various N/P ratios. ( C ) The cytotoxicities of Cs- g -PLLD or Cs- g -PLLD-FA in 143B cells were evaluated using the MTT assay. Note:  Error bars represent standard deviations calculated from three independent experiments. Abbreviations:  N/P, nitrogen/phosphorus; FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs- g -PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; siSCR, scrambled small interfering RNA; Lip, Lipofectamine 2000.
    Figure Legend Snippet: Electrophoretic migration of AEG-1 siRNA, Cs- g -PLLD/siAEG-1 ( A ), and Cs- g -PLLD-FA/siAEG-1 ( B ) complexes at various N/P ratios. ( C ) The cytotoxicities of Cs- g -PLLD or Cs- g -PLLD-FA in 143B cells were evaluated using the MTT assay. Note: Error bars represent standard deviations calculated from three independent experiments. Abbreviations: N/P, nitrogen/phosphorus; FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs- g -PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; siSCR, scrambled small interfering RNA; Lip, Lipofectamine 2000.

    Techniques Used: Migration, MTT Assay, Conjugation Assay, Modification, Small Interfering RNA

    ( A ) Fluorescence images under an inverted fluorescence microscope (×400 magnification) and ( B ) representative histograms of the percentage of fluorescent cells analyzed by flow cytometry for cy3-siRNA fluorescence 24 hours after the final transfection in 143B cells. Scale bar 50 μm. Abbreviations:  N/P, nitrogen/phosphorus; FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs- g -PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; Lip, Lipofectamine 2000.
    Figure Legend Snippet: ( A ) Fluorescence images under an inverted fluorescence microscope (×400 magnification) and ( B ) representative histograms of the percentage of fluorescent cells analyzed by flow cytometry for cy3-siRNA fluorescence 24 hours after the final transfection in 143B cells. Scale bar 50 μm. Abbreviations: N/P, nitrogen/phosphorus; FA, folic acid; PLLD, poly (L-lysine) dendrons; AEG-1, astrocyte elevated gene-1; Cs- g -PLLD-FA, a novel nanoscale polysaccharide derivative prepared by click conjugation of azido-modified chitosan with propargyl focal point PLLD and subsequent coupling with FA; Lip, Lipofectamine 2000.

    Techniques Used: Fluorescence, Microscopy, Flow Cytometry, Cytometry, Transfection, Conjugation Assay, Modification

    4) Product Images from "Effect of celastrol on toll-like receptor 4-mediated inflammatory response in free fatty acid-induced HepG2 cells"

    Article Title: Effect of celastrol on toll-like receptor 4-mediated inflammatory response in free fatty acid-induced HepG2 cells

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3775

    Effects of transfection with TLR4 siRNA in the HepG2 cells. In order to select the most effective TLR4 siRNA sequences, three FAM-labeled TLR4 siRNA constructs were transiently transfected into HepG2 cells using Lipofectamine ®  2000. After 24 h of transfection, the transfection efficiency was tested by a FAM-siRNA under a fluorescence microscope (magnification, x200). (A) Visualization of the cells under common light and FAM-fluorescence light. The fluorescent particles within the cells indicated that siRNA was transfected successfully into the HepG2 cells. (B) Relative mRNA and (C) relative protein expression levels of TLR4 were analyzed by reverse transcription-quantitative polymerase chain and western blotting, respectively. (D) FFA + negative control siRNA and (E) TLR4 siRNA-treated HepG2 cells stained by Oil red O (magnification, x400). Red arrows indicate lipid droplets. (F) Levels of intracellular triglycerides were analyzed by an enzymatic assay. Data are expressed as the mean ± standard error of the mean (n=6).  * P
    Figure Legend Snippet: Effects of transfection with TLR4 siRNA in the HepG2 cells. In order to select the most effective TLR4 siRNA sequences, three FAM-labeled TLR4 siRNA constructs were transiently transfected into HepG2 cells using Lipofectamine ® 2000. After 24 h of transfection, the transfection efficiency was tested by a FAM-siRNA under a fluorescence microscope (magnification, x200). (A) Visualization of the cells under common light and FAM-fluorescence light. The fluorescent particles within the cells indicated that siRNA was transfected successfully into the HepG2 cells. (B) Relative mRNA and (C) relative protein expression levels of TLR4 were analyzed by reverse transcription-quantitative polymerase chain and western blotting, respectively. (D) FFA + negative control siRNA and (E) TLR4 siRNA-treated HepG2 cells stained by Oil red O (magnification, x400). Red arrows indicate lipid droplets. (F) Levels of intracellular triglycerides were analyzed by an enzymatic assay. Data are expressed as the mean ± standard error of the mean (n=6). * P

    Techniques Used: Transfection, Labeling, Construct, Fluorescence, Microscopy, Expressing, Western Blot, Negative Control, Staining, Enzymatic Assay

    5) Product Images from "A Method to Culture GABAergic Interneurons Derived from the Medial Ganglionic Eminence"

    Article Title: A Method to Culture GABAergic Interneurons Derived from the Medial Ganglionic Eminence

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00423

    Setup of transfections of MGE-derived cells. (A,B) Test of different conditions of MGE–derived cells plated on 200 μg/ml PLL and 20 μg/ml LN, transfected at DIV1, and fixed at either DIV4 or DIV6 for immunofluorescence. After transfection cells were cultured without (A) or with 50 ng/ml BDNF (B) . (C) Quantification of the percentage of cells transfected at DIV1 with 0.1 μg of the indicated plasmid DNA and 0.5 μl of Lipofectamine-200, and fixed at different DIVs for immunofluorescence. Bars are means ±SEM of percentage of DAPI-positive cells expressing GFP. (D) Immunofluorescence of a culture transfected with GFP grown in the presence of BDNF, stained with Abs for GFP (green), for MAP2 (red), and with DAPI (blue). (E) DIV6 cells cotrasfected at DIV1 with 0.05 μg of each plasmid (total of 0.1 μg of plasmid DNA), to express GFP and HA-tagged guanine nucleotide exchange factor PIX.
    Figure Legend Snippet: Setup of transfections of MGE-derived cells. (A,B) Test of different conditions of MGE–derived cells plated on 200 μg/ml PLL and 20 μg/ml LN, transfected at DIV1, and fixed at either DIV4 or DIV6 for immunofluorescence. After transfection cells were cultured without (A) or with 50 ng/ml BDNF (B) . (C) Quantification of the percentage of cells transfected at DIV1 with 0.1 μg of the indicated plasmid DNA and 0.5 μl of Lipofectamine-200, and fixed at different DIVs for immunofluorescence. Bars are means ±SEM of percentage of DAPI-positive cells expressing GFP. (D) Immunofluorescence of a culture transfected with GFP grown in the presence of BDNF, stained with Abs for GFP (green), for MAP2 (red), and with DAPI (blue). (E) DIV6 cells cotrasfected at DIV1 with 0.05 μg of each plasmid (total of 0.1 μg of plasmid DNA), to express GFP and HA-tagged guanine nucleotide exchange factor PIX.

    Techniques Used: Transfection, Derivative Assay, Immunofluorescence, Cell Culture, Plasmid Preparation, Expressing, Staining

    6) Product Images from "IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice, et al. IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice"

    Article Title: IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice, et al. IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13610

    Knock‐down of AMPKα induces macrophage autophagy. Purified PMΦs from normal mice were transfected with pooled siRNAs targeting α‐subunit of AMPK or negative control (NC) siRNA using Lipofectamine 2000 (Lipo 2000) as described in Methods. 48 h later, (A) total AMPKα and phosphorylated AMPKα were detected by Western blotting. Blots shown are representative of three independent experiments. B‐D, cells were treated with PBS, SEA, IL‐7 or SEA plus IL‐7 for another 24 h. B and C, LC3II expression was detected by FCM. B, FCM plots are representative of experiments. C, Data were MFI ± SD of three independent experiments. D, Autophagosomes were detected by TEM. Data were means ± SD of 150 macrophages from three independent experiments. (* P ≤ .05, ** P ≤ .01, *** P ≤ .001)
    Figure Legend Snippet: Knock‐down of AMPKα induces macrophage autophagy. Purified PMΦs from normal mice were transfected with pooled siRNAs targeting α‐subunit of AMPK or negative control (NC) siRNA using Lipofectamine 2000 (Lipo 2000) as described in Methods. 48 h later, (A) total AMPKα and phosphorylated AMPKα were detected by Western blotting. Blots shown are representative of three independent experiments. B‐D, cells were treated with PBS, SEA, IL‐7 or SEA plus IL‐7 for another 24 h. B and C, LC3II expression was detected by FCM. B, FCM plots are representative of experiments. C, Data were MFI ± SD of three independent experiments. D, Autophagosomes were detected by TEM. Data were means ± SD of 150 macrophages from three independent experiments. (* P ≤ .05, ** P ≤ .01, *** P ≤ .001)

    Techniques Used: Purification, Mouse Assay, Transfection, Negative Control, Western Blot, Expressing, Transmission Electron Microscopy

    7) Product Images from "Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells"

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2018.00320

    mGluR4 activation inhibits the expression of cyclin D1 in LN229 cells.  (A)  LN229 cells were transfected with vehicle only, non-specific siRNA (siNC), and two mGluR4-targeted siRNAs (simGluR4-1 and simGluR4-2) using Lipofectamine 2000. mGluR4 protein levels were examined by western blot (WB). Samples isolated from cerebellar tissue (CBL) were used as a method control.  (B)  WB bands were quantified to generate the ratio of mGluR4 to β-actin for estimation of the downregulation of mGluR4 gene expression.  *** P
    Figure Legend Snippet: mGluR4 activation inhibits the expression of cyclin D1 in LN229 cells. (A) LN229 cells were transfected with vehicle only, non-specific siRNA (siNC), and two mGluR4-targeted siRNAs (simGluR4-1 and simGluR4-2) using Lipofectamine 2000. mGluR4 protein levels were examined by western blot (WB). Samples isolated from cerebellar tissue (CBL) were used as a method control. (B) WB bands were quantified to generate the ratio of mGluR4 to β-actin for estimation of the downregulation of mGluR4 gene expression. *** P

    Techniques Used: Activation Assay, Expressing, Transfection, Western Blot, Isolation

    8) Product Images from "Calbindin-D28k in the Brain Influences the Expression of Cellular Prion Protein"

    Article Title: Calbindin-D28k in the Brain Influences the Expression of Cellular Prion Protein

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/4670210

    Protein expression of PrP C  in GH3 cells and PC12 cells. Rat pituitary GH3 cells and pheochromocytoma PC12 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS at 37°C in 5% CO 2 , 95% air in a humidified cell incubator. To investigate the role of  CaBP-28k  in PC12 cells, cells were transfected with siRNA for  CaBP-28k  using Lipofectamine 2000. Expression of PrP C  protein was analyzed by Western blot as described in Experimental Procedures.
    Figure Legend Snippet: Protein expression of PrP C in GH3 cells and PC12 cells. Rat pituitary GH3 cells and pheochromocytoma PC12 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS at 37°C in 5% CO 2 , 95% air in a humidified cell incubator. To investigate the role of CaBP-28k in PC12 cells, cells were transfected with siRNA for CaBP-28k using Lipofectamine 2000. Expression of PrP C protein was analyzed by Western blot as described in Experimental Procedures.

    Techniques Used: Expressing, Cell Culture, Transfection, Western Blot

    9) Product Images from "Dopamine Receptor Subtypes Differentially Regulate Autophagy"

    Article Title: Dopamine Receptor Subtypes Differentially Regulate Autophagy

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19051540

    DRD1 and DRD5 knockdown promote autophagic flux. ( A ) DRD1 RNAi was combined with autophagy inhibitor Baf A1 (20 nM) for 2 h to detect the autophagic flux in HeLa cells stably expressing DRD1-GFP-3FLAG. ( B ) Total of 1 μg of MSCV-DRD1-GFP-3FLAG and MSCV-GFP-3FLAG plasmids were transfected into HeLa cells using lipofectamine 2000 for 48 h. ( C ) 0.2 or 0.5 μg MSCV-DRD1/DRD5-GFP-3FLAG or MSCV-GFP-3FLAG plasmid was transfected into 293T cells using lipofectamine 2000 for 48 h. ( D ) DRD5 RNAi was combined with autophagy inhibitors CQ (40 μM) for 2 h to detect the autophagic flux in HeLa cells stably expressing DRD5-GFP-3FLAG. The asterisk (*) indicates the nonspecific band. Experiments were repeated at least three times and representative Western blots are shown. Densitometric analysis was performed and quantification results were labeled below the corresponding blots or in separate panels. * p
    Figure Legend Snippet: DRD1 and DRD5 knockdown promote autophagic flux. ( A ) DRD1 RNAi was combined with autophagy inhibitor Baf A1 (20 nM) for 2 h to detect the autophagic flux in HeLa cells stably expressing DRD1-GFP-3FLAG. ( B ) Total of 1 μg of MSCV-DRD1-GFP-3FLAG and MSCV-GFP-3FLAG plasmids were transfected into HeLa cells using lipofectamine 2000 for 48 h. ( C ) 0.2 or 0.5 μg MSCV-DRD1/DRD5-GFP-3FLAG or MSCV-GFP-3FLAG plasmid was transfected into 293T cells using lipofectamine 2000 for 48 h. ( D ) DRD5 RNAi was combined with autophagy inhibitors CQ (40 μM) for 2 h to detect the autophagic flux in HeLa cells stably expressing DRD5-GFP-3FLAG. The asterisk (*) indicates the nonspecific band. Experiments were repeated at least three times and representative Western blots are shown. Densitometric analysis was performed and quantification results were labeled below the corresponding blots or in separate panels. * p

    Techniques Used: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Labeling

    10) Product Images from "Fabrication of Positively Charged Fluorescent Polymer Nanoparticles for Cell Imaging and Gene Delivery"

    Article Title: Fabrication of Positively Charged Fluorescent Polymer Nanoparticles for Cell Imaging and Gene Delivery

    Journal: Nanotheranostics

    doi: 10.7150/ntno.22988

    The gene expression results after co-cultured the nano-vector with Hek 293 cell for 36 h.  i ) EGFP+CPNPs,  ii ) DsRed+CPNPs,  iii ) EGFP+Lipofectamine 2000,  iv ) DsRed+Lipofectamine 2000. Pictures from left to right are the bright-field, fluorescence and merged microscopic images respectively.
    Figure Legend Snippet: The gene expression results after co-cultured the nano-vector with Hek 293 cell for 36 h. i ) EGFP+CPNPs, ii ) DsRed+CPNPs, iii ) EGFP+Lipofectamine 2000, iv ) DsRed+Lipofectamine 2000. Pictures from left to right are the bright-field, fluorescence and merged microscopic images respectively.

    Techniques Used: Expressing, Cell Culture, Plasmid Preparation, Fluorescence

    11) Product Images from "Interferon regulatory factor 4 negatively regulates the production of proinflammatory cytokines by macrophages in response to LPS"

    Article Title: Interferon regulatory factor 4 negatively regulates the production of proinflammatory cytokines by macrophages in response to LPS

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0504226102

    The levels of IRF-4 expression in macrophages affect the production of TNF-α. ( A ) Peritoneal macrophages were transfected with IRF-4-specific siRNA, control siRNA, or Lipofectamine 2000 only. Eight hours later, the expression of IRF-4 mRNA was determined by real-time RT-PCR. ( B ) Peritoneal macrophages were transfected with control siRNA (open bars) or IRF-4 specific siRNA (filled bars), cultured for 5 h, washed, and cultured in the presence of LPS for an additional 24 h. ( C ) Peritoneal macrophages transfected with control or IRF-4-specific siRNA were cultured with LPS (1 μg/ml) for the indicated period. The expression levels of TNF-α and IL-12 mRNA were determined by real-time RT-PCR analysis. ( D ) RAW264.7 cells were transfected with pcDNA3 or pcDNA3-mIRF-4. After5hof culture, cell lysate was separated by 12.5% SDS/PAGE. The blot was probed with anti-IRF-4 Ab, stripped, and reprobed with anti-Flag mAb. The same sample was separated by SDS/PAGE, blotted, and probed with anti-actin Ab. ( E ) RAW264.7 cells were transfected with pcDNA3 or pcDNA3-mIRF-4, cultured for 5 h, and stimulated with LPS for 8 h. ( F ) RAW-264.7 cells transfected with empty vector (pcDNA3) or with IRF-4 (pcDNA3-mIRF4) were stimulated with LPS. Cell lysate was separated by 12.5% SDS/PAGE. The blot was probed with anti-phospho-JNK Ab, stripped, and reprobed with anti-JNK Ab. The same blot was stripped and reprobed with anti-IRF-4 Ab.
    Figure Legend Snippet: The levels of IRF-4 expression in macrophages affect the production of TNF-α. ( A ) Peritoneal macrophages were transfected with IRF-4-specific siRNA, control siRNA, or Lipofectamine 2000 only. Eight hours later, the expression of IRF-4 mRNA was determined by real-time RT-PCR. ( B ) Peritoneal macrophages were transfected with control siRNA (open bars) or IRF-4 specific siRNA (filled bars), cultured for 5 h, washed, and cultured in the presence of LPS for an additional 24 h. ( C ) Peritoneal macrophages transfected with control or IRF-4-specific siRNA were cultured with LPS (1 μg/ml) for the indicated period. The expression levels of TNF-α and IL-12 mRNA were determined by real-time RT-PCR analysis. ( D ) RAW264.7 cells were transfected with pcDNA3 or pcDNA3-mIRF-4. After5hof culture, cell lysate was separated by 12.5% SDS/PAGE. The blot was probed with anti-IRF-4 Ab, stripped, and reprobed with anti-Flag mAb. The same sample was separated by SDS/PAGE, blotted, and probed with anti-actin Ab. ( E ) RAW264.7 cells were transfected with pcDNA3 or pcDNA3-mIRF-4, cultured for 5 h, and stimulated with LPS for 8 h. ( F ) RAW-264.7 cells transfected with empty vector (pcDNA3) or with IRF-4 (pcDNA3-mIRF4) were stimulated with LPS. Cell lysate was separated by 12.5% SDS/PAGE. The blot was probed with anti-phospho-JNK Ab, stripped, and reprobed with anti-JNK Ab. The same blot was stripped and reprobed with anti-IRF-4 Ab.

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Cell Culture, SDS Page, Plasmid Preparation

    12) Product Images from "Chimeric peptide nucleic acid compounds modulate splicing of the bcl-x gene in vitro and in vivo"

    Article Title: Chimeric peptide nucleic acid compounds modulate splicing of the bcl-x gene in vitro and in vivo

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki960

    The PNA-SR oligo specifically alters the bcl-xL to - xS ratio in vivo . ( A ) HeLa cells were transfected with Lipofectamine and the indicated amounts of PNA-(SR) 8 oligo, the PNA alone or the SR peptide. After 48 h, total RNA was isolated, reverse transcribed, and amplified by PCR. Splicing ratios and error bars have been obtained from three independent experiments. ( B ) Western blot of protein extracts derived from transfected HeLa cells. Cell lysates were analyzed with a bcl-x specific polyclonal antibody (upper panel) or a polyclonal antibody specific for hnRNP H/H′ as a control (lower panel). The asterisk symbol indicates a non-specific band recognized by the bcl-x antibody.
    Figure Legend Snippet: The PNA-SR oligo specifically alters the bcl-xL to - xS ratio in vivo . ( A ) HeLa cells were transfected with Lipofectamine and the indicated amounts of PNA-(SR) 8 oligo, the PNA alone or the SR peptide. After 48 h, total RNA was isolated, reverse transcribed, and amplified by PCR. Splicing ratios and error bars have been obtained from three independent experiments. ( B ) Western blot of protein extracts derived from transfected HeLa cells. Cell lysates were analyzed with a bcl-x specific polyclonal antibody (upper panel) or a polyclonal antibody specific for hnRNP H/H′ as a control (lower panel). The asterisk symbol indicates a non-specific band recognized by the bcl-x antibody.

    Techniques Used: In Vivo, Transfection, Isolation, Amplification, Polymerase Chain Reaction, Western Blot, Derivative Assay

    13) Product Images from "mTOR attenuates the inflammatory response in cardiomyocytes and prevents cardiac dysfunction in pathological hypertrophy"

    Article Title: mTOR attenuates the inflammatory response in cardiomyocytes and prevents cardiac dysfunction in pathological hypertrophy

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00338.2010

    mTOR overexpression in HL-1 cells. A : representative immunoblots of HA, mTOR, phospho-Akt, phospho-S6, total Akt, and total S6 in HL-1 cells transfected with mTOR. HL-1 cells were transfected with mock vector or mTOR using Lipofectamine 2000 as described
    Figure Legend Snippet: mTOR overexpression in HL-1 cells. A : representative immunoblots of HA, mTOR, phospho-Akt, phospho-S6, total Akt, and total S6 in HL-1 cells transfected with mTOR. HL-1 cells were transfected with mock vector or mTOR using Lipofectamine 2000 as described

    Techniques Used: Over Expression, Western Blot, Transfection, Plasmid Preparation

    14) Product Images from "Alteration of Golgi Structure in Senescent Cells and its Regulation by a G protein ? subunit"

    Article Title: Alteration of Golgi Structure in Senescent Cells and its Regulation by a G protein ? subunit

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2011.01.001

    Dispersal of trans Golgi and trans Golgi network (TGN) in senescent cells A. Dispersal of Golgi in stress induced senescence in HeLa cells . For trans Golgi labeling, HeLa cells were transfected with GalT- DsRed using lipofectamine 2000 (Invitrogen, Carlsbad, CA). The localization of GalT in live cells was monitored by epifluorescence microscopy. For trans Golgi network labeling, HeLa cells were grown on coverslips, treated for 24 hrs with BrdU to induce senescence, fixed and stained using the anti-TGN46 antibody (Sigma, St.Louis, MO). Top panels, basal cells, bottom panel, senescent cells (n > 3). B. Replicative senescence in IMR90 and WI-38 cells. Early (16-17 PDL) and late PDL (46 PDL) IMR90 and WI38 cells were fixed and stained for TGN46 distribution. Images were acquired using widefield microscopy. Top panels, early PDL cells, and bottom panels, late PDL cells. C. Bar graph depicting the number of cells with compact or dispersed TGN in late and early PDL IMR90 and WI38 cells. Average plot of two experiments. The percentage variability across the experiments was
    Figure Legend Snippet: Dispersal of trans Golgi and trans Golgi network (TGN) in senescent cells A. Dispersal of Golgi in stress induced senescence in HeLa cells . For trans Golgi labeling, HeLa cells were transfected with GalT- DsRed using lipofectamine 2000 (Invitrogen, Carlsbad, CA). The localization of GalT in live cells was monitored by epifluorescence microscopy. For trans Golgi network labeling, HeLa cells were grown on coverslips, treated for 24 hrs with BrdU to induce senescence, fixed and stained using the anti-TGN46 antibody (Sigma, St.Louis, MO). Top panels, basal cells, bottom panel, senescent cells (n > 3). B. Replicative senescence in IMR90 and WI-38 cells. Early (16-17 PDL) and late PDL (46 PDL) IMR90 and WI38 cells were fixed and stained for TGN46 distribution. Images were acquired using widefield microscopy. Top panels, early PDL cells, and bottom panels, late PDL cells. C. Bar graph depicting the number of cells with compact or dispersed TGN in late and early PDL IMR90 and WI38 cells. Average plot of two experiments. The percentage variability across the experiments was

    Techniques Used: Labeling, Transfection, Epifluorescence Microscopy, Staining, Microscopy

    15) Product Images from "Maitotoxin converts the plasmalemmal Ca2+ pump into a Ca2+-permeable nonselective cation channel"

    Article Title: Maitotoxin converts the plasmalemmal Ca2+ pump into a Ca2+-permeable nonselective cation channel

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00252.2009

    Effect of small interfering RNA (siRNA) on PMCA protein expression and on MTX-induced membrane currents.  Top:  HEK cells were transfected with either SMARTpool PMCA-siRNA or a nontargeting (NT) siRNA control (Dharmacon) using Lipofectamine 2000. Cells
    Figure Legend Snippet: Effect of small interfering RNA (siRNA) on PMCA protein expression and on MTX-induced membrane currents. Top: HEK cells were transfected with either SMARTpool PMCA-siRNA or a nontargeting (NT) siRNA control (Dharmacon) using Lipofectamine 2000. Cells

    Techniques Used: Small Interfering RNA, Expressing, Transfection

    16) Product Images from "BCYRN1, a c-MYC-activated long non-coding RNA, regulates cell metastasis of non-small-cell lung cancer"

    Article Title: BCYRN1, a c-MYC-activated long non-coding RNA, regulates cell metastasis of non-small-cell lung cancer

    Journal: Cancer Cell International

    doi: 10.1186/s12935-015-0183-3

    The RNA (A, C) and protein (B, D) expression levels of MMP9 and MMP13 were both downregulated by BCYRN1 inhibition, and upregulated by BCYRN1 mimics.  siRNA-NC: siRNA negative control; siRNA-1: siRNA-BCYRN1-1; siRNA-2: siRNA-BCYRN1-2; NC: negative control for BCYRN1 mimics, only treated with Lipofectamine 2000.  * p
    Figure Legend Snippet: The RNA (A, C) and protein (B, D) expression levels of MMP9 and MMP13 were both downregulated by BCYRN1 inhibition, and upregulated by BCYRN1 mimics. siRNA-NC: siRNA negative control; siRNA-1: siRNA-BCYRN1-1; siRNA-2: siRNA-BCYRN1-2; NC: negative control for BCYRN1 mimics, only treated with Lipofectamine 2000. * p

    Techniques Used: Expressing, Inhibition, Negative Control

    17) Product Images from "Inhibition of Hepatitis B Virus and Induction of Hepatoma Cell Apoptosis by ASGPR-Directed Delivery of shRNAs"

    Article Title: Inhibition of Hepatitis B Virus and Induction of Hepatoma Cell Apoptosis by ASGPR-Directed Delivery of shRNAs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046096

    The jetPEI-Hepatocyte reagent mediates specific shRNA delivery into HepG2.2.15 cells. ( A ) Representative images for EGFP expression in the cells that are transfected with pGenesil-1. Vehicle control, Lipofectamine 2000 (Lip2000) and jetPEI-Hepatocyte (JetPEI) reagents were used to mediate transfection. HepG2.2.15 and MDA-MD-231 cells were imaged by fluorescent microscopy at 24-h post-transfection. ( B ) Percentages of EGFP-positive cells detected by flow cytometry at 24-h post-transfection. HepG2.2.15 and MDA-MD-231 cells were transfected with pGenesil-scramble and pGenesil-siHBV4 respectively. Three reagents were used for transfection: vehicle control, Lip2000, and JetPEI. ( C ) The levels of the HBsAg protein in culture medium normalized to the level of untreated cells at 72 h post-transfection. HepG2.2.15 cells were transfected with pGenesil-scramble, pGenesil-siHBV4, and pGenesil-siSurvivin respectively using either Lip2000 or JetPEI. ( D ) Percentages of apoptotic cells detected by flow cytometry at 48 h post-transfection. HepG2.2.15 cells were transfected as in (C) (the left side of the composite figure). MDA-MB-231 and HepG2.2.15 cells were transfected with pGenesil-siSurvivin by JetPEI (the right side of the figure). Data in (B), (C), and (D) are shown as mean ± SD of three independent experiments (*: P
    Figure Legend Snippet: The jetPEI-Hepatocyte reagent mediates specific shRNA delivery into HepG2.2.15 cells. ( A ) Representative images for EGFP expression in the cells that are transfected with pGenesil-1. Vehicle control, Lipofectamine 2000 (Lip2000) and jetPEI-Hepatocyte (JetPEI) reagents were used to mediate transfection. HepG2.2.15 and MDA-MD-231 cells were imaged by fluorescent microscopy at 24-h post-transfection. ( B ) Percentages of EGFP-positive cells detected by flow cytometry at 24-h post-transfection. HepG2.2.15 and MDA-MD-231 cells were transfected with pGenesil-scramble and pGenesil-siHBV4 respectively. Three reagents were used for transfection: vehicle control, Lip2000, and JetPEI. ( C ) The levels of the HBsAg protein in culture medium normalized to the level of untreated cells at 72 h post-transfection. HepG2.2.15 cells were transfected with pGenesil-scramble, pGenesil-siHBV4, and pGenesil-siSurvivin respectively using either Lip2000 or JetPEI. ( D ) Percentages of apoptotic cells detected by flow cytometry at 48 h post-transfection. HepG2.2.15 cells were transfected as in (C) (the left side of the composite figure). MDA-MB-231 and HepG2.2.15 cells were transfected with pGenesil-siSurvivin by JetPEI (the right side of the figure). Data in (B), (C), and (D) are shown as mean ± SD of three independent experiments (*: P

    Techniques Used: shRNA, Expressing, Transfection, Multiple Displacement Amplification, Microscopy, Flow Cytometry, Cytometry

    18) Product Images from "Dual engagement of the NLRP3 and AIM2 inflammasomes by plasmodial-derived hemozoin and DNA during malaria"

    Article Title: Dual engagement of the NLRP3 and AIM2 inflammasomes by plasmodial-derived hemozoin and DNA during malaria

    Journal: Cell reports

    doi: 10.1016/j.celrep.2013.12.014

    nHz and sHz/ Pf gDNA complex induce IL-1β production and pyroptosis both in vitro and in vivo via NLRP3 and AIM2 (a) Macrophages stably expressing ASC-CFP were left untreated or treated with nHz (100µM). The formation of ASC pyroptosomes was visualized using confocal microscopy. Scale bar: 15µm (left) and 5µm (right) (b) Primary wt, Nlrp3 −/− , Aim2 +/+ , Aim2 −/− and Nlrp3 −/− , Aim2 −/− BMDMs were incubated for 12 h with 15 µM and 30 µM of nHz, nigericin (10µM) or transfected with 1.5µg/ml poly (dAdT) using Lipofectamine. Cell culture supernatants were assessed for IL-1β release by ELISA (c) Primary wt, Nlrp3 −/− , Aim2 +/+ , Aim2 −/− , and Nlrp3 −/− , Aim2 −/− BMDMs were incubated for 12 h with 20 µM, 40 µM and 80 µM of nHz or transfected with Poly (dAdT). Cell survival was measured using calcein AM. Medium was set as 100%. Data are mean cytokine levels ± SD of triplicate determinations and are representative of 3 independent experiments (d) wt, Nlrp3 −/− , Aim2 +/+ , Aim2 −/− and Nlrp3 −/− Aim2 −/− mice were i.v. injected in the tail vein with endotoxin-free PBS or sHz/ Pf gDNA (1mg sHz, 15µg Pf gDNA). After 12 h livers were dissected and homogenized. Each lane corresponds to one mouse. (e) wt (n=12), Nlrp3 −/− (n=16), Aim2 +/+ (n=9), Aim2 −/− (n=14), and Nlrp3 −/− Aim2 −/− (n=6) mice were intraperitonally injected with sHz/ Pf gDNA (1mg sHz, 15µg Pf gDNA) or PBS. After 15 h, peritoneal cells were harvested, counted and FACS analysis was performed using GR-1 antibody. Basal neutrophil influx in PBS injected mice was subtracted to determine total number of neutrophils recruited to the peritoneal cavity. Student’s t-test was used to calculate P values (*p
    Figure Legend Snippet: nHz and sHz/ Pf gDNA complex induce IL-1β production and pyroptosis both in vitro and in vivo via NLRP3 and AIM2 (a) Macrophages stably expressing ASC-CFP were left untreated or treated with nHz (100µM). The formation of ASC pyroptosomes was visualized using confocal microscopy. Scale bar: 15µm (left) and 5µm (right) (b) Primary wt, Nlrp3 −/− , Aim2 +/+ , Aim2 −/− and Nlrp3 −/− , Aim2 −/− BMDMs were incubated for 12 h with 15 µM and 30 µM of nHz, nigericin (10µM) or transfected with 1.5µg/ml poly (dAdT) using Lipofectamine. Cell culture supernatants were assessed for IL-1β release by ELISA (c) Primary wt, Nlrp3 −/− , Aim2 +/+ , Aim2 −/− , and Nlrp3 −/− , Aim2 −/− BMDMs were incubated for 12 h with 20 µM, 40 µM and 80 µM of nHz or transfected with Poly (dAdT). Cell survival was measured using calcein AM. Medium was set as 100%. Data are mean cytokine levels ± SD of triplicate determinations and are representative of 3 independent experiments (d) wt, Nlrp3 −/− , Aim2 +/+ , Aim2 −/− and Nlrp3 −/− Aim2 −/− mice were i.v. injected in the tail vein with endotoxin-free PBS or sHz/ Pf gDNA (1mg sHz, 15µg Pf gDNA). After 12 h livers were dissected and homogenized. Each lane corresponds to one mouse. (e) wt (n=12), Nlrp3 −/− (n=16), Aim2 +/+ (n=9), Aim2 −/− (n=14), and Nlrp3 −/− Aim2 −/− (n=6) mice were intraperitonally injected with sHz/ Pf gDNA (1mg sHz, 15µg Pf gDNA) or PBS. After 15 h, peritoneal cells were harvested, counted and FACS analysis was performed using GR-1 antibody. Basal neutrophil influx in PBS injected mice was subtracted to determine total number of neutrophils recruited to the peritoneal cavity. Student’s t-test was used to calculate P values (*p

    Techniques Used: In Vitro, In Vivo, Stable Transfection, Expressing, Confocal Microscopy, Incubation, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, FACS

    Pf gDNA induces IL-1β through activation of AIM2 inflammasome (a) 50 and 100 ng/ml of Pf gDNA (3D7) were transfected using lipofectamine into LPS primed (100ng/ml × 2h) immortalized murine wt, Nlrp3 −/− , Asc −/− and casp1 −/− BMDMs. After 12h, IL-1β was measured in the culture supernatants by ELISA. (b) 100ng/ml of gDNA from different strains of Pf (Dd2, T9/94, HB3-B2) was transfected using lipofectamine into LPS-primed primary Aim2 +/+ and Aim2 −/− BMDMs. Cell culture supernatants were used to measure IL-1β using ELISA. Data are presented as mean ± SD of triplicates and are representative of 3 independent experiments. (c) Confocal microscopy of LPS primed ASC-CFP cells left untrasfected or transfected with 100ng/ml Syto60-labeled Pf gDNA. Scale bar: 20µm (top), 5µm (bottom) (d) Confocal microscopy of LPS primed AIM2-citrine macrophages left untransfected or transfected with 100ng/ml DAPI-labeled Pf gDNA, DAPI-labeled poly (dAdT) using lipofectamine or just incubated with sHz. Scale bar (from top to bottom): 15µm, 5µm, 15µm and 15µm. (e) Confocal microscopy of NLRP3-citrine macrophages untreated or treated with sHz/CpG (5µg/ml), sHz/ Pf gDNA (4µg/ml), nigericin or transfected with 100ng/ml Pf .
    Figure Legend Snippet: Pf gDNA induces IL-1β through activation of AIM2 inflammasome (a) 50 and 100 ng/ml of Pf gDNA (3D7) were transfected using lipofectamine into LPS primed (100ng/ml × 2h) immortalized murine wt, Nlrp3 −/− , Asc −/− and casp1 −/− BMDMs. After 12h, IL-1β was measured in the culture supernatants by ELISA. (b) 100ng/ml of gDNA from different strains of Pf (Dd2, T9/94, HB3-B2) was transfected using lipofectamine into LPS-primed primary Aim2 +/+ and Aim2 −/− BMDMs. Cell culture supernatants were used to measure IL-1β using ELISA. Data are presented as mean ± SD of triplicates and are representative of 3 independent experiments. (c) Confocal microscopy of LPS primed ASC-CFP cells left untrasfected or transfected with 100ng/ml Syto60-labeled Pf gDNA. Scale bar: 20µm (top), 5µm (bottom) (d) Confocal microscopy of LPS primed AIM2-citrine macrophages left untransfected or transfected with 100ng/ml DAPI-labeled Pf gDNA, DAPI-labeled poly (dAdT) using lipofectamine or just incubated with sHz. Scale bar (from top to bottom): 15µm, 5µm, 15µm and 15µm. (e) Confocal microscopy of NLRP3-citrine macrophages untreated or treated with sHz/CpG (5µg/ml), sHz/ Pf gDNA (4µg/ml), nigericin or transfected with 100ng/ml Pf .

    Techniques Used: Activation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Confocal Microscopy, Labeling, Incubation

    19) Product Images from "Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo"

    Article Title: Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20051229

    Cytokine secretion from human intestinal epithelial LoVo cells.  LoVo cells were treated with 2 μg/ml of each ligand, except 200 ng/ml for AcMTP and 10 ng/ml TNFα. The levels of IL-8 (A) and CXCL1 (E) were determined by ELISA. (B) LoVo cells were treated with 20 μg/ml KF1B, 5 μg/ml AcMTP, 1 μM CpG, 5 μg/ml polyIC, and 5 μg/ml  E. coli  O55:B5 LPS. The levels of IL-8 in medium at the indicated times were determined by ELISA. (C) SW620 cells were treated with 5 μg/ml of the indicated ligands. The secretion levels of IL-8 were determined by ELISA. (D) LoVo cells were treated transfected with NF-κB–dependent and –independent reporter by Lipofectamine 2000. 8 h after transfection, cells were treated with 2,000 ng/ml KF1B, iE-DAP, or control  meso DAP or left untreated (−). (F) The mRNA levels of CD83, NFKBIA, IL-8, and CXCL1 from cells treated with 2 μg/ml KF1B (closed bars) for 60 min or left untreated (open bars) were determined by real-time PCR analysis. (G) CD83 in lysates from LoVo cells treated with 2,000 ng/ml KF1B for 24 h was detected by immunoblotting with anti-CD83 antibody. The results shown are given as means ± SD of triplicate cultures and were representative of five experiments.
    Figure Legend Snippet: Cytokine secretion from human intestinal epithelial LoVo cells. LoVo cells were treated with 2 μg/ml of each ligand, except 200 ng/ml for AcMTP and 10 ng/ml TNFα. The levels of IL-8 (A) and CXCL1 (E) were determined by ELISA. (B) LoVo cells were treated with 20 μg/ml KF1B, 5 μg/ml AcMTP, 1 μM CpG, 5 μg/ml polyIC, and 5 μg/ml E. coli O55:B5 LPS. The levels of IL-8 in medium at the indicated times were determined by ELISA. (C) SW620 cells were treated with 5 μg/ml of the indicated ligands. The secretion levels of IL-8 were determined by ELISA. (D) LoVo cells were treated transfected with NF-κB–dependent and –independent reporter by Lipofectamine 2000. 8 h after transfection, cells were treated with 2,000 ng/ml KF1B, iE-DAP, or control meso DAP or left untreated (−). (F) The mRNA levels of CD83, NFKBIA, IL-8, and CXCL1 from cells treated with 2 μg/ml KF1B (closed bars) for 60 min or left untreated (open bars) were determined by real-time PCR analysis. (G) CD83 in lysates from LoVo cells treated with 2,000 ng/ml KF1B for 24 h was detected by immunoblotting with anti-CD83 antibody. The results shown are given as means ± SD of triplicate cultures and were representative of five experiments.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Real-time Polymerase Chain Reaction

    20) Product Images from "Translation, but not transfection limits clinically relevant, exogenous mRNA based induction of alpha-4 integrin expression on human mesenchymal stem cells"

    Article Title: Translation, but not transfection limits clinically relevant, exogenous mRNA based induction of alpha-4 integrin expression on human mesenchymal stem cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01304-3

    SSB protein binding assay in vitro . Electrophoresis after SSB protein to ITGA4-mRNA in vitro binding assay: 1) 1 kb DNA ladder, 2) ITGA4-mRNA, 3) ITGA4-mRNA/SSB, 4) ITGA4-mRNA/SSB/Lipofectamine ® 2000, 5) SSB/Lipofectamine ® 2000, 6) SSB, 7) Lipofectamine®2000.
    Figure Legend Snippet: SSB protein binding assay in vitro . Electrophoresis after SSB protein to ITGA4-mRNA in vitro binding assay: 1) 1 kb DNA ladder, 2) ITGA4-mRNA, 3) ITGA4-mRNA/SSB, 4) ITGA4-mRNA/SSB/Lipofectamine ® 2000, 5) SSB/Lipofectamine ® 2000, 6) SSB, 7) Lipofectamine®2000.

    Techniques Used: Protein Binding, In Vitro, Electrophoresis, Binding Assay

    ITGA4 protein synthesis after ITGA4-mRNA/SSB transfection. Immunocytochemistry study for assessment of the ITGA4 protein presence after ITGA4-mRNA/SSB transfection using Lipofectamine ® 2000. Scale bars separate for HEK293 and MSC cells located in the respective left upper images of the panel indicate 100 µm.
    Figure Legend Snippet: ITGA4 protein synthesis after ITGA4-mRNA/SSB transfection. Immunocytochemistry study for assessment of the ITGA4 protein presence after ITGA4-mRNA/SSB transfection using Lipofectamine ® 2000. Scale bars separate for HEK293 and MSC cells located in the respective left upper images of the panel indicate 100 µm.

    Techniques Used: Transfection, Immunocytochemistry

    21) Product Images from "A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells"

    Article Title: A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0182941

    Comparison of major commercially available transfection reagents using described flow cytometric method that quantifies transfection efficiency. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and different commercially available transfection reagents as described in Methods. Gating on viable cells was performed as shown in Fig 1 . Each experiment was performed in triplicates and six independent times (6 per plasmid and 12 in total) with specific (un)labelled nucleic acids. Data in each experiment were normalized by the average of the experimental control (standard transfected nucleic acids) in each experiment and were then pooled together. This approach increases statistical power while taking into consideration the inherent differences in transfection efficiency among different transfection methods (different chemical transfection reagents and plasmids). The non-parametric statistical Mann-Whitney test was used for comparisons between groups. Median and interquartile range (IQR) are shown. The transfection efficiency was assessed by both amount of labelled DNA (A) and protein (B). The use of TransIT-X2 and Jet Prime gave the best (and comparable) transfection efficiencies, whereas the efficiency was reduced with Lipofectamine 2000 and Fugene HD. There was a major decrease in the amount of detectable labelled DNA with Lipofectamine 2000 and Fugene HD.
    Figure Legend Snippet: Comparison of major commercially available transfection reagents using described flow cytometric method that quantifies transfection efficiency. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and different commercially available transfection reagents as described in Methods. Gating on viable cells was performed as shown in Fig 1 . Each experiment was performed in triplicates and six independent times (6 per plasmid and 12 in total) with specific (un)labelled nucleic acids. Data in each experiment were normalized by the average of the experimental control (standard transfected nucleic acids) in each experiment and were then pooled together. This approach increases statistical power while taking into consideration the inherent differences in transfection efficiency among different transfection methods (different chemical transfection reagents and plasmids). The non-parametric statistical Mann-Whitney test was used for comparisons between groups. Median and interquartile range (IQR) are shown. The transfection efficiency was assessed by both amount of labelled DNA (A) and protein (B). The use of TransIT-X2 and Jet Prime gave the best (and comparable) transfection efficiencies, whereas the efficiency was reduced with Lipofectamine 2000 and Fugene HD. There was a major decrease in the amount of detectable labelled DNA with Lipofectamine 2000 and Fugene HD.

    Techniques Used: Transfection, Flow Cytometry, Cytometry, Labeling, Plasmid Preparation, MANN-WHITNEY

    22) Product Images from "Luteolin Inhibits Tumorigenesis and Induces Apoptosis of Non-Small Cell Lung Cancer Cells via Regulation of MicroRNA-34a-5p"

    Article Title: Luteolin Inhibits Tumorigenesis and Induces Apoptosis of Non-Small Cell Lung Cancer Cells via Regulation of MicroRNA-34a-5p

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19020447

    MDM4 is a direct target of miR-34a-5p in A549 and H460 cells. ( A ) The sequence of miR-34a-5p and its binding sites in the 3′-UTR of MDM4 mRNA; The red letters stand for complementary base pairing between miRNA-34a-5p and 3′-UTR of MDM4 mRNA; ( B , C ) Luciferase activity was measured in A549 and H460 cells with a dual-luciferase reporter assay. The MDM4-3′-UTR or the mutant MDM4-3′-UTR vectors and the miR-34a-5p mimics or negative controls were co-transfected into cells with Lipofectamine 2000. The relative luciferase activity was normalized to the Renilla luciferase activity. The results are expressed as means ± SD of three independent experiments. *  p
    Figure Legend Snippet: MDM4 is a direct target of miR-34a-5p in A549 and H460 cells. ( A ) The sequence of miR-34a-5p and its binding sites in the 3′-UTR of MDM4 mRNA; The red letters stand for complementary base pairing between miRNA-34a-5p and 3′-UTR of MDM4 mRNA; ( B , C ) Luciferase activity was measured in A549 and H460 cells with a dual-luciferase reporter assay. The MDM4-3′-UTR or the mutant MDM4-3′-UTR vectors and the miR-34a-5p mimics or negative controls were co-transfected into cells with Lipofectamine 2000. The relative luciferase activity was normalized to the Renilla luciferase activity. The results are expressed as means ± SD of three independent experiments. * p

    Techniques Used: Sequencing, Binding Assay, Luciferase, Activity Assay, Reporter Assay, Mutagenesis, Transfection

    23) Product Images from "Quantum dots to monitor RNAi delivery and improve gene silencing"

    Article Title: Quantum dots to monitor RNAi delivery and improve gene silencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni188

    Optimization of QD concentration for siRNA tracking. Lmna siRNA (100 nM) and 1, 2.5, 5 or 10 µg QD were co-transfected into murine fibroblasts and the cells FACS-sorted for the low 10% (L + ) and high 10% (H + ) of intracellular fluorescence distribution. ( A ) Protein expression of sorted cells assayed by western blot, β-actin loading control. Unsorted, lipofectamine only control (U) represented 100% lamin a/c protein expression. ( B ) Western blot band densitometry analysis of L + and H + bands shows an optimum QD concentration for obtaining high-efficiency silencing. ( C ) Band density difference (L + minus H + ) reveals an optimum QD concentration for sorting most efficiently silenced from least efficiently silenced subpopulations.
    Figure Legend Snippet: Optimization of QD concentration for siRNA tracking. Lmna siRNA (100 nM) and 1, 2.5, 5 or 10 µg QD were co-transfected into murine fibroblasts and the cells FACS-sorted for the low 10% (L + ) and high 10% (H + ) of intracellular fluorescence distribution. ( A ) Protein expression of sorted cells assayed by western blot, β-actin loading control. Unsorted, lipofectamine only control (U) represented 100% lamin a/c protein expression. ( B ) Western blot band densitometry analysis of L + and H + bands shows an optimum QD concentration for obtaining high-efficiency silencing. ( C ) Band density difference (L + minus H + ) reveals an optimum QD concentration for sorting most efficiently silenced from least efficiently silenced subpopulations.

    Techniques Used: Concentration Assay, Transfection, FACS, Fluorescence, Expressing, Western Blot

    24) Product Images from "Down-Regulation of Gli Transcription Factor Leads to the Inhibition of Migration and Invasion of Ovarian Cancer Cells via Integrin ?4-Mediated FAK Signaling"

    Article Title: Down-Regulation of Gli Transcription Factor Leads to the Inhibition of Migration and Invasion of Ovarian Cancer Cells via Integrin ?4-Mediated FAK Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088386

    ITGB4 mediates Shh-induced ovarian cancer cell invasion. ( A, B ) Stimulation of N-Shh up-regulates ITGB4 expression. SKOV3 cells were incubated in the presence or absence of N-Shh (0.5 µg/ml) for 24 hr. Western blots were then performed for the indicated antibodies. Protein expression was quantified by Image J and normalized to GAPDH. ( C, D ) Inhibition of Gli1 down-regulates ITGB4 expression. SKOV3 cells were treated with GANT61 or DMSO for 48 hr. Western blot was then performed with the indicated antibodies. Protein expression was quantified by Image J and normalized to GAPDH. ( E, F ) Blockade of ITGB4 inhibits cell invasion in SKOV3 cells. SKOV3 cells grown in low serum (2% FBS) growth medium were seeded into the upper chambers. The low serum growth medium containing N-Shh (0.5 µg/ml) together with anti-ITGB4 antibody (0.2 µg/ml) or control IgG was added to the lower chamber. After 24 hr of incubation, cells that invaded the lower surface of the insert were stained with crystal violet and counted by microscopy. ( G ) Gli1 miRNAi inhibits the expression of ITGB4 in SKOV3 cells. SKOV3 cells were transiently transfected with vehicle (lipofectamine alone), control miRNAi and Gli1 miRNAis (miR-Gli1-720 and miR-Gli1-1863), respectively. Western blot showed that Gli1 miRNAi inhibited the expression of ITGB4 genes. ( H ) Silencing Gli1 expression impairs Shh-induced ovarian cancer cell invasion. SKOV3 cells were transfected with Gli1 miRNAi or control miRNAi for 24 hr followed by stimulation with N-Shh (0.5 µg/ml) or control medium for another 24 hr. Cell invasion was measured as described in Methods . ( I, J ) Silencing ITGB4 expression impairs Shh-induced ovarian cancer cell invasion. SKOV3 cells were transfected with ITGB4 miRNAi constructs (miR-ITGB4-2255 and miR-ITGB4-4027) or control miRNAi for 24 hr followed by stimulation with N-Shh (0.5 µg/ml) or control medium for another 24 hr. Western blot showed that ITGB4 miRNAis inhibited the expression of ITGB4 gene ( I ). Cell invasion was measured as described in Methods ( J ). The data are expressed as mean ± SD for experiments performed three times. **, P
    Figure Legend Snippet: ITGB4 mediates Shh-induced ovarian cancer cell invasion. ( A, B ) Stimulation of N-Shh up-regulates ITGB4 expression. SKOV3 cells were incubated in the presence or absence of N-Shh (0.5 µg/ml) for 24 hr. Western blots were then performed for the indicated antibodies. Protein expression was quantified by Image J and normalized to GAPDH. ( C, D ) Inhibition of Gli1 down-regulates ITGB4 expression. SKOV3 cells were treated with GANT61 or DMSO for 48 hr. Western blot was then performed with the indicated antibodies. Protein expression was quantified by Image J and normalized to GAPDH. ( E, F ) Blockade of ITGB4 inhibits cell invasion in SKOV3 cells. SKOV3 cells grown in low serum (2% FBS) growth medium were seeded into the upper chambers. The low serum growth medium containing N-Shh (0.5 µg/ml) together with anti-ITGB4 antibody (0.2 µg/ml) or control IgG was added to the lower chamber. After 24 hr of incubation, cells that invaded the lower surface of the insert were stained with crystal violet and counted by microscopy. ( G ) Gli1 miRNAi inhibits the expression of ITGB4 in SKOV3 cells. SKOV3 cells were transiently transfected with vehicle (lipofectamine alone), control miRNAi and Gli1 miRNAis (miR-Gli1-720 and miR-Gli1-1863), respectively. Western blot showed that Gli1 miRNAi inhibited the expression of ITGB4 genes. ( H ) Silencing Gli1 expression impairs Shh-induced ovarian cancer cell invasion. SKOV3 cells were transfected with Gli1 miRNAi or control miRNAi for 24 hr followed by stimulation with N-Shh (0.5 µg/ml) or control medium for another 24 hr. Cell invasion was measured as described in Methods . ( I, J ) Silencing ITGB4 expression impairs Shh-induced ovarian cancer cell invasion. SKOV3 cells were transfected with ITGB4 miRNAi constructs (miR-ITGB4-2255 and miR-ITGB4-4027) or control miRNAi for 24 hr followed by stimulation with N-Shh (0.5 µg/ml) or control medium for another 24 hr. Western blot showed that ITGB4 miRNAis inhibited the expression of ITGB4 gene ( I ). Cell invasion was measured as described in Methods ( J ). The data are expressed as mean ± SD for experiments performed three times. **, P

    Techniques Used: Expressing, Incubation, Western Blot, Inhibition, Staining, Microscopy, Transfection, Construct

    25) Product Images from "β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding"

    Article Title: β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00296.2017

    β-PIX silencing on β-PIX/GIT1/Rac1 interactions and cell migration.  A : representative immunoblots of β-PIX, GIT1 and Rac1. IEC-Cdx2L1 cells were transfected with control siRNA (C-siRNA) or siβ-PIX by LipofectAMINE 2000, and whole cell lysates were harvested 48 h thereafter. Levels of β-PIX, GIT1, and Rac1 proteins were measured by Western immunoblot analysis, and GAPDH immunoblotting was performed as an internal control for equal loading. Three separate experiments were performed that showed similar results.  B : quantitative analysis of β-PIX immunoblots by densitometry that were corrected for GAPDH loading from cells described above. Values are means ± SE. *  P
    Figure Legend Snippet: β-PIX silencing on β-PIX/GIT1/Rac1 interactions and cell migration. A : representative immunoblots of β-PIX, GIT1 and Rac1. IEC-Cdx2L1 cells were transfected with control siRNA (C-siRNA) or siβ-PIX by LipofectAMINE 2000, and whole cell lysates were harvested 48 h thereafter. Levels of β-PIX, GIT1, and Rac1 proteins were measured by Western immunoblot analysis, and GAPDH immunoblotting was performed as an internal control for equal loading. Three separate experiments were performed that showed similar results. B : quantitative analysis of β-PIX immunoblots by densitometry that were corrected for GAPDH loading from cells described above. Values are means ± SE. * P

    Techniques Used: Migration, Western Blot, Transfection

    Ectopic overexpression of β-PIX on β-PIX/GIT1/Rac1 association and cell migration. A : representative immunoblots of β-PIX and GIT1. Caco-2 cells were transfected with β-PIX expression vector or empty vector (Null) by LipofectAMINE 2000 for 48 and 72 h, and levels of β-PIX and GIT1 proteins were examined by Western immunoblotting analysis. Three separate experiments were performed that showed similar results. B : quantitative analysis of β-PIX immunoblots by densitometry that were corrected for GAPDH loading from cells described above. Values are means ± SE. * P
    Figure Legend Snippet: Ectopic overexpression of β-PIX on β-PIX/GIT1/Rac1 association and cell migration. A : representative immunoblots of β-PIX and GIT1. Caco-2 cells were transfected with β-PIX expression vector or empty vector (Null) by LipofectAMINE 2000 for 48 and 72 h, and levels of β-PIX and GIT1 proteins were examined by Western immunoblotting analysis. Three separate experiments were performed that showed similar results. B : quantitative analysis of β-PIX immunoblots by densitometry that were corrected for GAPDH loading from cells described above. Values are means ± SE. * P

    Techniques Used: Over Expression, Migration, Western Blot, Transfection, Expressing, Plasmid Preparation

    26) Product Images from "Selective recognition of c-MYC Pu22 G-quadruplex by a fluorescent probe"

    Article Title: Selective recognition of c-MYC Pu22 G-quadruplex by a fluorescent probe

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz059

    Fluorescence images of A549 cells transfected with 40 pmol c-MYC Pu22 DNA G-quadruplex forming oligonucleotide labelled with 5′-Cy5 using lipofectamine 2000 and then incubated with 5 μM 9Cl for 2 hours successively. ( A ) Fluorescence signal collected between 655–755 nm at λ ex = 635 nm, ( B ) fluorescence signal collected between 425–470 nm at λ ex = 405 nm, ( C ) fluorescence signal collected between 500 and 545 nm at λ ex = 405 nm, ( D ) merged image of (A) and (C), ( E ) merged images of (A), (B), (C) and the bright field. Scale bar, 20 μm.
    Figure Legend Snippet: Fluorescence images of A549 cells transfected with 40 pmol c-MYC Pu22 DNA G-quadruplex forming oligonucleotide labelled with 5′-Cy5 using lipofectamine 2000 and then incubated with 5 μM 9Cl for 2 hours successively. ( A ) Fluorescence signal collected between 655–755 nm at λ ex = 635 nm, ( B ) fluorescence signal collected between 425–470 nm at λ ex = 405 nm, ( C ) fluorescence signal collected between 500 and 545 nm at λ ex = 405 nm, ( D ) merged image of (A) and (C), ( E ) merged images of (A), (B), (C) and the bright field. Scale bar, 20 μm.

    Techniques Used: Fluorescence, Transfection, Incubation

    27) Product Images from "Silibinin down-regulates FAT10 and modulate TNF-α/IFN-γ-induced chromosomal instability and apoptosis sensitivity"

    Article Title: Silibinin down-regulates FAT10 and modulate TNF-α/IFN-γ-induced chromosomal instability and apoptosis sensitivity

    Journal: Biology Open

    doi: 10.1242/bio.011189

    Silibinin inhibits TNF-α/IFN-γ (TI)-induced endogenous FAT10 expression through NF-κB signaling pathway.  (A) Seven predicted binding sites of NF-κB were found within 2.5 kb upstream of FAT10 promoter region by GenomatixMatInspector Version 8.0 ( http://www.genomatix.de ). (B) p65 plays a role in modulating FAT10 expression induced by TI. Cells were electroporated with control siRNA (ctr) or siRNAs against p65 (p65) and grown for 24 h. They were then cultured with or without TI for 8 h before harvest. Inhibition of NF-κB subunit p65 expression by its specific siRNA and FAT10 expression were confirmed by western blot. (C) Silibinin inhibits TI-induced FAT10 mRNA levels in a dose dependent manner. Real-time reverse transcription (RT)-PCR was performed to measure the levels of FAT10 mRNA. (D1) Silibinin inhibits TI-induced FAT10 promoter and (D2) Silibinin inhibits NF-κB activities. FAT10-promoter driven β-galactosidase reporter and the NF-κB-SEAP constructs were co-transfected into HepG2 cells using Lipofectamine 2000. 36 h after transfection, cells were treated with TNF-α (50 ng/ml) and IFN-γ (50 U/ml), in the presence or absence of 100 µM silibinin. Nine hours later, cells were harvested and FAT10-promoter driven β-galactosidase reporter and NF-κB driven SEAP activities were determined and normalized against GFP expression and total protein content. All data are shown as mean±s.e. (standard error). ** P
    Figure Legend Snippet: Silibinin inhibits TNF-α/IFN-γ (TI)-induced endogenous FAT10 expression through NF-κB signaling pathway. (A) Seven predicted binding sites of NF-κB were found within 2.5 kb upstream of FAT10 promoter region by GenomatixMatInspector Version 8.0 ( http://www.genomatix.de ). (B) p65 plays a role in modulating FAT10 expression induced by TI. Cells were electroporated with control siRNA (ctr) or siRNAs against p65 (p65) and grown for 24 h. They were then cultured with or without TI for 8 h before harvest. Inhibition of NF-κB subunit p65 expression by its specific siRNA and FAT10 expression were confirmed by western blot. (C) Silibinin inhibits TI-induced FAT10 mRNA levels in a dose dependent manner. Real-time reverse transcription (RT)-PCR was performed to measure the levels of FAT10 mRNA. (D1) Silibinin inhibits TI-induced FAT10 promoter and (D2) Silibinin inhibits NF-κB activities. FAT10-promoter driven β-galactosidase reporter and the NF-κB-SEAP constructs were co-transfected into HepG2 cells using Lipofectamine 2000. 36 h after transfection, cells were treated with TNF-α (50 ng/ml) and IFN-γ (50 U/ml), in the presence or absence of 100 µM silibinin. Nine hours later, cells were harvested and FAT10-promoter driven β-galactosidase reporter and NF-κB driven SEAP activities were determined and normalized against GFP expression and total protein content. All data are shown as mean±s.e. (standard error). ** P

    Techniques Used: Expressing, Binding Assay, Cell Culture, Inhibition, Western Blot, Reverse Transcription Polymerase Chain Reaction, Construct, Transfection

    28) Product Images from "Cx43 Inhibition Attenuates Sepsis-Induced Intestinal Injury via Downregulating ROS Transfer and the Activation of the JNK1/Sirt1/FoxO3a Signaling Pathway"

    Article Title: Cx43 Inhibition Attenuates Sepsis-Induced Intestinal Injury via Downregulating ROS Transfer and the Activation of the JNK1/Sirt1/FoxO3a Signaling Pathway

    Journal: Mediators of Inflammation

    doi: 10.1155/2019/7854389

    FoxO3a directly regulated Bim and Puma expression. (a) FoxO3a siRNAs (FoxO3a-siRNA1 and FoxO3a-siRNA2) attenuated FoxO3a expression in IEC-6 cells. (b) The increase of Bim luciferase activity was attenuated by FoxO3a siRNA transient transfection into IEC-6 cells with Lipofectamine 2000. (c) IEC-6 cells were pretreated with FoxO3a siRNAs (FoxO3a-siRNA1 and FoxO3a-siRNA2) and then subjected to LPS (10  μ g/ml) for 24 hours. ChIP analyses were performed with antibodies against FoxO3a and primers for the Bim promoter regions. (d) The increase of Puma luciferase activity was attenuated by FoxO3a siRNA transient transfection into IEC-6 cells with Lipofectamine 2000. (c) IEC-6 cells were pretreated with FoxO3a siRNAs (FoxO3a-siRNA1 and FoxO3a-siRNA2) and then subjected to LPS (10  μ g/ml) for 24 hours. ChIP analyses were performed with antibodies against FoxO3a and primers for the Puma promoter regions. Data are shown as mean ± SD,  n  = 3;  ∗ p
    Figure Legend Snippet: FoxO3a directly regulated Bim and Puma expression. (a) FoxO3a siRNAs (FoxO3a-siRNA1 and FoxO3a-siRNA2) attenuated FoxO3a expression in IEC-6 cells. (b) The increase of Bim luciferase activity was attenuated by FoxO3a siRNA transient transfection into IEC-6 cells with Lipofectamine 2000. (c) IEC-6 cells were pretreated with FoxO3a siRNAs (FoxO3a-siRNA1 and FoxO3a-siRNA2) and then subjected to LPS (10  μ g/ml) for 24 hours. ChIP analyses were performed with antibodies against FoxO3a and primers for the Bim promoter regions. (d) The increase of Puma luciferase activity was attenuated by FoxO3a siRNA transient transfection into IEC-6 cells with Lipofectamine 2000. (c) IEC-6 cells were pretreated with FoxO3a siRNAs (FoxO3a-siRNA1 and FoxO3a-siRNA2) and then subjected to LPS (10  μ g/ml) for 24 hours. ChIP analyses were performed with antibodies against FoxO3a and primers for the Puma promoter regions. Data are shown as mean ± SD, n = 3; ∗ p

    Techniques Used: Expressing, Luciferase, Activity Assay, Transfection, Chromatin Immunoprecipitation

    29) Product Images from "Effective Gene Silencing by Multilayered siRNA Coated Gold Nanoparticles**"

    Article Title: Effective Gene Silencing by Multilayered siRNA Coated Gold Nanoparticles**

    Journal: Small (Weinheim an Der Bergstrasse, Germany)

    doi: 10.1002/smll.201001314

    Cytotoxicity of multilayer sRAuNPs. Cell viability was evaluated by MTT assay after transfection with multilayer sRAuNPs or Lipofectamine 2000 in MDA-MB231-luc2 (a) and LNCaP-luc2 (b) cell lines. Results are representative of three independent experiments.
    Figure Legend Snippet: Cytotoxicity of multilayer sRAuNPs. Cell viability was evaluated by MTT assay after transfection with multilayer sRAuNPs or Lipofectamine 2000 in MDA-MB231-luc2 (a) and LNCaP-luc2 (b) cell lines. Results are representative of three independent experiments.

    Techniques Used: MTT Assay, Transfection, Multiple Displacement Amplification

    Gene silencing effect of multilayer sRAuNP in MDA-MB231-luc2 cell line. Luminescence signal in MDA-MB231-luc2 cells after incubation with different sRAuNPs or Lipofectamine 2000 was evaluated by IVIS 200 (a). Value of luminescence intensity (photon/sec)
    Figure Legend Snippet: Gene silencing effect of multilayer sRAuNP in MDA-MB231-luc2 cell line. Luminescence signal in MDA-MB231-luc2 cells after incubation with different sRAuNPs or Lipofectamine 2000 was evaluated by IVIS 200 (a). Value of luminescence intensity (photon/sec)

    Techniques Used: Multiple Displacement Amplification, Incubation, Size-exclusion Chromatography

    30) Product Images from "JNK signaling is required for the MIP-1α-associated regulation of Kupffer cells in the heat stroke response"

    Article Title: JNK signaling is required for the MIP-1α-associated regulation of Kupffer cells in the heat stroke response

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.6922

    Expression of MIP-1α positively regulates KC inflammatory function in HS. (A) The livers of the experimental animals were prepared following the induction of  HS for 1–5 h. MIP-1α (red) expression in the KCs (green) detected by immunofluorescence revealed an increase in expression during the time course in the HS group compared with the control group. (B) KCs and liver extracts were prepared following the induction of HS for 1–5 h. The amount of MIP-1α that was up-regulated  in vitro  and  in vivo  was investigated by western blot analysis. (C) MIP-1α expression was reduced using specifically targeted siRNAs in cultured KCs transfected using Lipofectamine ®  2000. (D) IFN-γ, IL-1β, and TNF-α concentration in the medium of cultured KCs decreased in the MIP-1α-silenced HS group compared with the HS group detected by ELISA. *P
    Figure Legend Snippet: Expression of MIP-1α positively regulates KC inflammatory function in HS. (A) The livers of the experimental animals were prepared following the induction of HS for 1–5 h. MIP-1α (red) expression in the KCs (green) detected by immunofluorescence revealed an increase in expression during the time course in the HS group compared with the control group. (B) KCs and liver extracts were prepared following the induction of HS for 1–5 h. The amount of MIP-1α that was up-regulated in vitro and in vivo was investigated by western blot analysis. (C) MIP-1α expression was reduced using specifically targeted siRNAs in cultured KCs transfected using Lipofectamine ® 2000. (D) IFN-γ, IL-1β, and TNF-α concentration in the medium of cultured KCs decreased in the MIP-1α-silenced HS group compared with the HS group detected by ELISA. *P

    Techniques Used: Expressing, Immunofluorescence, In Vitro, In Vivo, Western Blot, Cell Culture, Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay

    31) Product Images from "Modeling ERBB receptor-regulated G1/S transition to find novel targets for de novo trastuzumab resistance"

    Article Title: Modeling ERBB receptor-regulated G1/S transition to find novel targets for de novo trastuzumab resistance

    Journal: BMC Systems Biology

    doi: 10.1186/1752-0509-3-1

    Proof of principle experiments for the determination of G1/S transition point in trastuzumab resistant HCC1954 cells . A. Western blots showing the expression and activation of key G1/S proteins. Cells were treated either only with Lipofectamine (MOCK) or with 20 nM CDK4 siRNA for 24 hours. Then, cells were synchronized for 24 hours and subsequently stimulated with 25 ng/ml EGF for 6, 12, 18 or 24 hours. Cell lysates were applied to immunoblotting. B. Reverse Phase Protein Array (RPPA) showing the phosphorylation state of pRB protein (Ser 807/811). The same lysates (from A) were applied to RPPA. The upper panel shows the read-out of antibody signal at near infra-red range for phospho-pRB antibody with four replicates. The lower panel shows the graphical representation of phospho-pRB antibody signal intensity for two different conditions and for four time points. Signals were normalized to 0 hour MOCK sample.
    Figure Legend Snippet: Proof of principle experiments for the determination of G1/S transition point in trastuzumab resistant HCC1954 cells . A. Western blots showing the expression and activation of key G1/S proteins. Cells were treated either only with Lipofectamine (MOCK) or with 20 nM CDK4 siRNA for 24 hours. Then, cells were synchronized for 24 hours and subsequently stimulated with 25 ng/ml EGF for 6, 12, 18 or 24 hours. Cell lysates were applied to immunoblotting. B. Reverse Phase Protein Array (RPPA) showing the phosphorylation state of pRB protein (Ser 807/811). The same lysates (from A) were applied to RPPA. The upper panel shows the read-out of antibody signal at near infra-red range for phospho-pRB antibody with four replicates. The lower panel shows the graphical representation of phospho-pRB antibody signal intensity for two different conditions and for four time points. Signals were normalized to 0 hour MOCK sample.

    Techniques Used: Western Blot, Expressing, Activation Assay, Protein Array

    Determination of knockdown efficiency and specificity of siRNAs in the HCC1954 cell system . A. qRT-PCR results showing the knockdown efficiency of 20 nM pools of four siRNAs for each gene in the network (50 nM siRNA for ESR1 ). ACTB and HPRT1 were used as house-keeping controls. MOCK stands for the samples which were treated only with Lipofectamine transfection reagent. B. Western blots for the determination of knockdowns for the network elements at protein level. Since dimers of ERBB family members are accepted as functional units, we used combinatorial RNAi (knockdown of two genes simultaneously) to produce knockdowns of such dimers. C. qRT-PCR results showing the knockdown efficiency and effect of one member of ERBB receptor family siRNA on the other two members of the family. Both pools of four individual siRNAs per gene and only one individual siRNA per gene were used. The concentration of siRNAs was 20 nM. ACTB was used as house-keeping control. D. Western blots showing the knockdown efficiency and cross reactivity of siRNAs at protein level. β-actin was taken as loading control.
    Figure Legend Snippet: Determination of knockdown efficiency and specificity of siRNAs in the HCC1954 cell system . A. qRT-PCR results showing the knockdown efficiency of 20 nM pools of four siRNAs for each gene in the network (50 nM siRNA for ESR1 ). ACTB and HPRT1 were used as house-keeping controls. MOCK stands for the samples which were treated only with Lipofectamine transfection reagent. B. Western blots for the determination of knockdowns for the network elements at protein level. Since dimers of ERBB family members are accepted as functional units, we used combinatorial RNAi (knockdown of two genes simultaneously) to produce knockdowns of such dimers. C. qRT-PCR results showing the knockdown efficiency and effect of one member of ERBB receptor family siRNA on the other two members of the family. Both pools of four individual siRNAs per gene and only one individual siRNA per gene were used. The concentration of siRNAs was 20 nM. ACTB was used as house-keeping control. D. Western blots showing the knockdown efficiency and cross reactivity of siRNAs at protein level. β-actin was taken as loading control.

    Techniques Used: Quantitative RT-PCR, Transfection, Western Blot, Functional Assay, Concentration Assay

    32) Product Images from "Target Genes of Neuron-Restrictive Silencer Factor Are Abnormally Up-Regulated in Human Myotilinopathy"

    Article Title: Target Genes of Neuron-Restrictive Silencer Factor Are Abnormally Up-Regulated in Human Myotilinopathy

    Journal:

    doi: 10.2353/ajpath.2007.070520

    NRSF/REST overexpression reduces endogenous levels of neuronal α-internexin in human SH-SY5Y cells.  A:  One μg of REEX1 vector was transfected in SH-SY5Y cells using Lipofectamine 2000. NRSF/REST protein levels were increased after 48 hours
    Figure Legend Snippet: NRSF/REST overexpression reduces endogenous levels of neuronal α-internexin in human SH-SY5Y cells. A: One μg of REEX1 vector was transfected in SH-SY5Y cells using Lipofectamine 2000. NRSF/REST protein levels were increased after 48 hours

    Techniques Used: Over Expression, Plasmid Preparation, Transfection

    NRSF/REST overexpression reduces α-internexin, SNAP25, synaptophysin, and UCHL1 mRNA levels in the DMS53 cell line. A: One μg of REEX1 vector, which encodes human full-length NRSF cDNA, was transfected in DMS53 cells using Lipofectamine
    Figure Legend Snippet: NRSF/REST overexpression reduces α-internexin, SNAP25, synaptophysin, and UCHL1 mRNA levels in the DMS53 cell line. A: One μg of REEX1 vector, which encodes human full-length NRSF cDNA, was transfected in DMS53 cells using Lipofectamine

    Techniques Used: Over Expression, Plasmid Preparation, Transfection

    33) Product Images from "Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair"

    Article Title: Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S71586

    Optimization of Arg-G4-based gene silencing. Notes: ( A ) Arg-G4-siRNA transfection efficiency in MSCs at various N/P ratios using Lipofectamine 2000 as a control. ( B ) Cell viability in MSCs during transfection at various N/P ratios using Lipofectamine 2000 as a control. ( C ) Arg-G4-siRNA transfection efficiency in MSCs using various siRNA concentrations. ( D ) Cell viability in MSCs during transfection using various siRNA concentrations. * P
    Figure Legend Snippet: Optimization of Arg-G4-based gene silencing. Notes: ( A ) Arg-G4-siRNA transfection efficiency in MSCs at various N/P ratios using Lipofectamine 2000 as a control. ( B ) Cell viability in MSCs during transfection at various N/P ratios using Lipofectamine 2000 as a control. ( C ) Arg-G4-siRNA transfection efficiency in MSCs using various siRNA concentrations. ( D ) Cell viability in MSCs during transfection using various siRNA concentrations. * P

    Techniques Used: Transfection

    34) Product Images from "Activation of type I interferon antiviral response in human neural stem cells"

    Article Title: Activation of type I interferon antiviral response in human neural stem cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-019-1521-5

    Poly(I:C) transfection upregulates mRNA expression of IFN-β and λ1 in hNSCs. a RT-qPCR analysis of IFN-β, α, and λ1 expression in poly(I:C)-transfected hNSCs (1 μg). The experiments were triplicated, and the error bars represented the standard deviation (SD). b Human NSCs were transfected with 0.2, 1, and 2 μg of poly(I:C) by Lipofectamine 2000 (LF2K), and the expression of IFN-β and IFN-λ1 was analyzed by RT-qPCR. The experiments were performed in triplicate, and the error bars represented the SD. c The supernatants of human NSCs transfected with 1 μg poly(I:C) were harvested at 6, 12, and 24 h post-transfection, and the protein expression of IFN-β was determined using ELISA. The experiments were performed in triplicate, and the error bars represented the SD. Student’s t test was used for statistical analysis. * p
    Figure Legend Snippet: Poly(I:C) transfection upregulates mRNA expression of IFN-β and λ1 in hNSCs. a RT-qPCR analysis of IFN-β, α, and λ1 expression in poly(I:C)-transfected hNSCs (1 μg). The experiments were triplicated, and the error bars represented the standard deviation (SD). b Human NSCs were transfected with 0.2, 1, and 2 μg of poly(I:C) by Lipofectamine 2000 (LF2K), and the expression of IFN-β and IFN-λ1 was analyzed by RT-qPCR. The experiments were performed in triplicate, and the error bars represented the SD. c The supernatants of human NSCs transfected with 1 μg poly(I:C) were harvested at 6, 12, and 24 h post-transfection, and the protein expression of IFN-β was determined using ELISA. The experiments were performed in triplicate, and the error bars represented the SD. Student’s t test was used for statistical analysis. * p

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Standard Deviation, Enzyme-linked Immunosorbent Assay

    RIG-I and MDA5 agonists stimulate the expression of IFN-β in hNSCs. a , b Human NSCs were transfected with 5′pppRNA or poly(I:C) HMW. Total RNA was collected at 12 and 24 h post-transfection, and RT-qPCR assay was performed to examine the relative amount of IFN-β mRNA ( a ). The expression of phospho-IRF3 and total IRF3 was detected through a western blot assay. β-actin was used as an internal control. ( b ). c Human NSCs were transfected with siRNA specific for RIG-I or MDA5 by Lipofectamine RNAiMAX 2000 and then transfected with 1 μg 5′pppRNA for 24 h. RT-qPCR analysis was performed to detect the expression of IFN-β transcripts while western blot was applied to confirm the knockdown efficiency. d Human NSCs were transfected with siRNA targeting RIG-I or MDA5 and then transfected with 1 μg of poly(I:C) HMW for 24 h. RT-qPCR analysis was performed to detect the expression of IFN-β transcripts while western blot was applied to confirm the knockdown efficiency. The experiments were performed in triplicate, and the error bars represented the SD. Student’s t test was used for statistical analysis. * p
    Figure Legend Snippet: RIG-I and MDA5 agonists stimulate the expression of IFN-β in hNSCs. a , b Human NSCs were transfected with 5′pppRNA or poly(I:C) HMW. Total RNA was collected at 12 and 24 h post-transfection, and RT-qPCR assay was performed to examine the relative amount of IFN-β mRNA ( a ). The expression of phospho-IRF3 and total IRF3 was detected through a western blot assay. β-actin was used as an internal control. ( b ). c Human NSCs were transfected with siRNA specific for RIG-I or MDA5 by Lipofectamine RNAiMAX 2000 and then transfected with 1 μg 5′pppRNA for 24 h. RT-qPCR analysis was performed to detect the expression of IFN-β transcripts while western blot was applied to confirm the knockdown efficiency. d Human NSCs were transfected with siRNA targeting RIG-I or MDA5 and then transfected with 1 μg of poly(I:C) HMW for 24 h. RT-qPCR analysis was performed to detect the expression of IFN-β transcripts while western blot was applied to confirm the knockdown efficiency. The experiments were performed in triplicate, and the error bars represented the SD. Student’s t test was used for statistical analysis. * p

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Western Blot

    35) Product Images from "Mitochondrial-targeted Aryl Hydrocarbon Receptor and the Impact of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Cellular Respiration and the Mitochondrial Proteome"

    Article Title: Mitochondrial-targeted Aryl Hydrocarbon Receptor and the Impact of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Cellular Respiration and the Mitochondrial Proteome

    Journal: Toxicology and applied pharmacology

    doi: 10.1016/j.taap.2016.04.005

    The effect of AIP knockdown on the amount of AHR in cellular fractions of hepa1c1c7 cells siRNA was used to knockdown AIP in Hepa1c1c7 cells. Western blot analysis for AIP and AHR expression was performed from whole cell lysate, and nuclear, cytosolic, and mitochondrial fractions. α-tubulin was used as a loading control for the whole cell lysate, histone H3 (H3) was a loading control for the nuclear fraction, lactate dehydrogenase (LDH) was a loading control for the cytosolic fraction, and cytochrome c oxidase subunit IV (COX4) was a loading control for the mitochondrial fraction. (A) Results shown are representative of 3 independent experiments. Lane 1: lipofectamine treatment (Lipomock), lane 2: 10 nM Silencer® Select Negative Control #1 siRNA (siCtrl), lane 3: 10 nM siRNA1 for AIP (siAIP1), lane4: 10 nM siRNA2 for AIP (siAIP2). (B) Densitometry was determined with a Fuji Image analyzer. The levels of expressions of AIP and AHR protein were normalized to each loading control. The normalized protein expression levels were compared with each other by re-normalization with the protein expression from which cells were affected by lipofectamine only (Lipomock). The bars represent mean ± the standard errors (n=3).
    Figure Legend Snippet: The effect of AIP knockdown on the amount of AHR in cellular fractions of hepa1c1c7 cells siRNA was used to knockdown AIP in Hepa1c1c7 cells. Western blot analysis for AIP and AHR expression was performed from whole cell lysate, and nuclear, cytosolic, and mitochondrial fractions. α-tubulin was used as a loading control for the whole cell lysate, histone H3 (H3) was a loading control for the nuclear fraction, lactate dehydrogenase (LDH) was a loading control for the cytosolic fraction, and cytochrome c oxidase subunit IV (COX4) was a loading control for the mitochondrial fraction. (A) Results shown are representative of 3 independent experiments. Lane 1: lipofectamine treatment (Lipomock), lane 2: 10 nM Silencer® Select Negative Control #1 siRNA (siCtrl), lane 3: 10 nM siRNA1 for AIP (siAIP1), lane4: 10 nM siRNA2 for AIP (siAIP2). (B) Densitometry was determined with a Fuji Image analyzer. The levels of expressions of AIP and AHR protein were normalized to each loading control. The normalized protein expression levels were compared with each other by re-normalization with the protein expression from which cells were affected by lipofectamine only (Lipomock). The bars represent mean ± the standard errors (n=3).

    Techniques Used: Western Blot, Expressing, Negative Control

    The effect of TOMM20 knockdown on the amount of AHR protein in cellular fractions of hepa1c1c7 cells siRNA was used to knockdown TOMM20 in Hepa1c1c7 cells. Western blot analysis for TOMM20 and AHR expression was performed from whole cell lysate, and nuclear, cytosolic and mitochondrial fractions. α-tubulin was used as a loading control for the whole cell lysate, histone H3 (H3) was a loading control for the nuclear fraction, lactate dehydrogenase (LDH) was a loading control for the cytosolic fraction and ATP5α was a loading control for the mitochondrial fraction. (A) Results shown are representative of three independent experiments. Lane 1: lipofectamine treatment (Lipomock), lane 2: 10 nM nontargeting siRNA (siCtrl), lane 3: 10 nM siRNA for TOMM20 (siTomm20). (B and C) Densitometry was determined with a Fuji Image analyzer. The expressions of TOMM20 and AHR protein were normalized to each loading control. The normalized protein expression levels were compared to each other by re-normalization with the protein expression from which cells were affected by lipofectamine only (Lipomock). The bars represent mean ± the standard errors (n=3).
    Figure Legend Snippet: The effect of TOMM20 knockdown on the amount of AHR protein in cellular fractions of hepa1c1c7 cells siRNA was used to knockdown TOMM20 in Hepa1c1c7 cells. Western blot analysis for TOMM20 and AHR expression was performed from whole cell lysate, and nuclear, cytosolic and mitochondrial fractions. α-tubulin was used as a loading control for the whole cell lysate, histone H3 (H3) was a loading control for the nuclear fraction, lactate dehydrogenase (LDH) was a loading control for the cytosolic fraction and ATP5α was a loading control for the mitochondrial fraction. (A) Results shown are representative of three independent experiments. Lane 1: lipofectamine treatment (Lipomock), lane 2: 10 nM nontargeting siRNA (siCtrl), lane 3: 10 nM siRNA for TOMM20 (siTomm20). (B and C) Densitometry was determined with a Fuji Image analyzer. The expressions of TOMM20 and AHR protein were normalized to each loading control. The normalized protein expression levels were compared to each other by re-normalization with the protein expression from which cells were affected by lipofectamine only (Lipomock). The bars represent mean ± the standard errors (n=3).

    Techniques Used: Western Blot, Expressing

    36) Product Images from "Synthesis, characterization and function of an RNA-based transfection reagent"

    Article Title: Synthesis, characterization and function of an RNA-based transfection reagent

    Journal: Current protocols in nucleic acid chemistry

    doi: 10.1002/cpnc.51

    Efficiency of 2′-OMeUtaPS and dTtaPS at inducing the excision of exon 23 from the  mdx  mouse dystrophin pre-mRNA upon transfection of the PMO or PNA sequence  14  or  15 , respectively, in  mdx  mouse myotubes. The concentration of 2′-OMeUtaPS or dTtaPS is kept at 2 μM in serum containing medium. Lipofectamine ® 2000 (LF) is used as a transfection reagent in serum free-medium at a concentration recommended by the supplier. Total RNA is extracted from transfected myotubes and amplified by nested RT-PCR using appropriate sets of DNA primers (Basic protocol 6). Two major secondary PCR products have been separated by electrophoresis on a 1.5% agarose gel; the larger 633 bp and shorter 420 bp PCR products correspond to the unspliced and correctly spliced pre-mRNA exon 23, respectively. SM = size marker.
    Figure Legend Snippet: Efficiency of 2′-OMeUtaPS and dTtaPS at inducing the excision of exon 23 from the mdx mouse dystrophin pre-mRNA upon transfection of the PMO or PNA sequence 14 or 15 , respectively, in mdx mouse myotubes. The concentration of 2′-OMeUtaPS or dTtaPS is kept at 2 μM in serum containing medium. Lipofectamine ® 2000 (LF) is used as a transfection reagent in serum free-medium at a concentration recommended by the supplier. Total RNA is extracted from transfected myotubes and amplified by nested RT-PCR using appropriate sets of DNA primers (Basic protocol 6). Two major secondary PCR products have been separated by electrophoresis on a 1.5% agarose gel; the larger 633 bp and shorter 420 bp PCR products correspond to the unspliced and correctly spliced pre-mRNA exon 23, respectively. SM = size marker.

    Techniques Used: Transfection, Sequencing, Concentration Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Marker

    37) Product Images from "Effect of the miR-96-5p inhibitor and mimic on the migration and invasion of the SW480-7 colorectal cancer cell line"

    Article Title: Effect of the miR-96-5p inhibitor and mimic on the migration and invasion of the SW480-7 colorectal cancer cell line

    Journal: Oncology Letters

    doi: 10.3892/ol.2019.10492

    Effects of miRNA-96-5p inhibitor on cell viability of (A) SW480, (B) SW620 and (C) HCT116. The values of cell viability were normalized with that of the corresponding negative control group. Untreated, only medium treated cells; LF, only Lipofectamine 2000 with medium treated cells; HiPerFect, only HiPerFect with medium treated cells; negative control, transfection with miR inhibitor control and cultured with medium; miR-96-5p, transfection with miR-96 inhibitor and cultured with medium. Data represent the mean ± SD of three independent experiments, each performed in triplicate.
    Figure Legend Snippet: Effects of miRNA-96-5p inhibitor on cell viability of (A) SW480, (B) SW620 and (C) HCT116. The values of cell viability were normalized with that of the corresponding negative control group. Untreated, only medium treated cells; LF, only Lipofectamine 2000 with medium treated cells; HiPerFect, only HiPerFect with medium treated cells; negative control, transfection with miR inhibitor control and cultured with medium; miR-96-5p, transfection with miR-96 inhibitor and cultured with medium. Data represent the mean ± SD of three independent experiments, each performed in triplicate.

    Techniques Used: Negative Control, Transfection, Cell Culture

    38) Product Images from "Tau reduction in the presence of amyloid-β prevents tau pathology and neuronal death in vivo"

    Article Title: Tau reduction in the presence of amyloid-β prevents tau pathology and neuronal death in vivo

    Journal: Brain

    doi: 10.1093/brain/awy117

    Human tau reduction decreases tau seeding activity despite amyloid-β presence. ( A ) A total of 0.1ug of total brain lysate per well was added to the HEK-TauRD CFP/YFP tau bioactivity sensor cells with Lipofectamine® and one well collected every 3 h for 24 h to measure the amount of aggregation—read out as FRET activity—over time for each sample. Integrated FRET Density was calculated for all samples by multiplying the per cent FRET positive cells by the median fluorescence intensity of the FRET positive population. Both the rTg4510 DOX (blue asterisk) and APP × rTg4510 DOX (green asterisk) samples exhibited less tau seeding activity than their naïve genotype controls. At the final 24 h point, a significant difference between the rTg4510 DOX and APP × rTg4510 DOX groups emerged (black asterisk). Treatment effect F (3,29) = 5.961, P = 0.0027. ( B ) Representative images of non-transduced cells (−), non-transgenic (NT), and APP/PS1 (APP) treated cells at the final 24 h collection point, all showing no aggregation. ( C ) Representative images of both rTg4510 and APP × rTg4510 genotypes and each treatment across all time points collected. Two-way repeated measures ANOVA, Sidak post hoc multiple comparisons. * P
    Figure Legend Snippet: Human tau reduction decreases tau seeding activity despite amyloid-β presence. ( A ) A total of 0.1ug of total brain lysate per well was added to the HEK-TauRD CFP/YFP tau bioactivity sensor cells with Lipofectamine® and one well collected every 3 h for 24 h to measure the amount of aggregation—read out as FRET activity—over time for each sample. Integrated FRET Density was calculated for all samples by multiplying the per cent FRET positive cells by the median fluorescence intensity of the FRET positive population. Both the rTg4510 DOX (blue asterisk) and APP × rTg4510 DOX (green asterisk) samples exhibited less tau seeding activity than their naïve genotype controls. At the final 24 h point, a significant difference between the rTg4510 DOX and APP × rTg4510 DOX groups emerged (black asterisk). Treatment effect F (3,29) = 5.961, P = 0.0027. ( B ) Representative images of non-transduced cells (−), non-transgenic (NT), and APP/PS1 (APP) treated cells at the final 24 h collection point, all showing no aggregation. ( C ) Representative images of both rTg4510 and APP × rTg4510 genotypes and each treatment across all time points collected. Two-way repeated measures ANOVA, Sidak post hoc multiple comparisons. * P

    Techniques Used: Activity Assay, Fluorescence, Transgenic Assay

    39) Product Images from "Widespread Effects of Chemokine 3’-UTRs on mRNA Degradation and Protein Production in Human Cells"

    Article Title: Widespread Effects of Chemokine 3’-UTRs on mRNA Degradation and Protein Production in Human Cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1800114

    A let-7 target and an ARE in the CCL3 3’-UTR co-regulate gene expression. ( A ) CCL3 3’-UTR contains a conserved let-7 target and an ARE. The vertical bars represent conservation scores from PhyloP. The let-7 target is predicted by TargetScan; the ARE (based on ARED) contains a typical ARE motif (solid underline) and two non-typical ARE motifs (broken underline). ( B ) Mutagenesis of the let-7 target and the ARE resulted in the reduction of inhibitory activity of CCL3 3’-UTR in BEAS-2B cells. WT: CCL3 3’-UTR; ARE-del, deletion of the ARE in CCL3 3’-UTR; Let7-scr, scrambled seed sequence binding site in CCL3 3’-UTR; ARE-del Let7-scr: mutation of both the ARE and the let-7 target. ( C ) The let-7 inhibitor abrogated the inhibitory effect of the let-7 target in CCL3 3’-UTR. Lipo: negative control transfection with lipofectamine; AntiLet7, let-7 inhibitor. ( D ) Scrambling the let-7 target seed sequence in CCL3 3’-UTR stabilized mRNA. **, p
    Figure Legend Snippet: A let-7 target and an ARE in the CCL3 3’-UTR co-regulate gene expression. ( A ) CCL3 3’-UTR contains a conserved let-7 target and an ARE. The vertical bars represent conservation scores from PhyloP. The let-7 target is predicted by TargetScan; the ARE (based on ARED) contains a typical ARE motif (solid underline) and two non-typical ARE motifs (broken underline). ( B ) Mutagenesis of the let-7 target and the ARE resulted in the reduction of inhibitory activity of CCL3 3’-UTR in BEAS-2B cells. WT: CCL3 3’-UTR; ARE-del, deletion of the ARE in CCL3 3’-UTR; Let7-scr, scrambled seed sequence binding site in CCL3 3’-UTR; ARE-del Let7-scr: mutation of both the ARE and the let-7 target. ( C ) The let-7 inhibitor abrogated the inhibitory effect of the let-7 target in CCL3 3’-UTR. Lipo: negative control transfection with lipofectamine; AntiLet7, let-7 inhibitor. ( D ) Scrambling the let-7 target seed sequence in CCL3 3’-UTR stabilized mRNA. **, p

    Techniques Used: Expressing, Mutagenesis, Activity Assay, Sequencing, Binding Assay, Negative Control, Transfection

    40) Product Images from "HIV-1 Vif N-terminal Motif is required for recruitment of Cul5 to Suppress APOBEC3"

    Article Title: HIV-1 Vif N-terminal Motif is required for recruitment of Cul5 to Suppress APOBEC3

    Journal: Retrovirology

    doi: 10.1186/1742-4690-11-4

    Vif N-terminal mutants localize to cytoplasm, but are inefficient at restoring HIV infectivity. HA-tagged Vif wild-type and mutant proteins along with CBF-β were overexpressed in HEK 293 T cells. Two days post-transfection, cells were lysed and cleared lysate was mixed with anti-HA matrix affinity beads for 4-8 hrs. Incubated beads were washed several times followed by elution of bound proteins. A) Select Vif N-terminal mutants that do not efficiently degrade A3G and A3F have a reduced ability to co-precipitate Cul5; however, CBF-β and Elo B/C can still bind Vif. B) Plasmids (Vif-YFP 2 ug and CBF-β 0.5 ug) were transfected into 293 T cells using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. Cells were visualized at 25°C using a Zeiss LSM510-Meta confocal imaging system. Imaging demonstrates that the Vif double mutant V25/H27A and single mutant H108A localize to the cytoplasm of the cell similar to wild-type. C) Vif wild-type and mutant containing virus were produced and used to infect MAGI cells. Infected cells were stained using X-gal. The histogram demonstrates that Cul5-binding deficient Vif mutants were inefficient at restoring HIV infectivity in the presence of A3G. Error bars represent the standard error from triplicate experiments. Capsid p24 levels are shown in the western blot.
    Figure Legend Snippet: Vif N-terminal mutants localize to cytoplasm, but are inefficient at restoring HIV infectivity. HA-tagged Vif wild-type and mutant proteins along with CBF-β were overexpressed in HEK 293 T cells. Two days post-transfection, cells were lysed and cleared lysate was mixed with anti-HA matrix affinity beads for 4-8 hrs. Incubated beads were washed several times followed by elution of bound proteins. A) Select Vif N-terminal mutants that do not efficiently degrade A3G and A3F have a reduced ability to co-precipitate Cul5; however, CBF-β and Elo B/C can still bind Vif. B) Plasmids (Vif-YFP 2 ug and CBF-β 0.5 ug) were transfected into 293 T cells using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. Cells were visualized at 25°C using a Zeiss LSM510-Meta confocal imaging system. Imaging demonstrates that the Vif double mutant V25/H27A and single mutant H108A localize to the cytoplasm of the cell similar to wild-type. C) Vif wild-type and mutant containing virus were produced and used to infect MAGI cells. Infected cells were stained using X-gal. The histogram demonstrates that Cul5-binding deficient Vif mutants were inefficient at restoring HIV infectivity in the presence of A3G. Error bars represent the standard error from triplicate experiments. Capsid p24 levels are shown in the western blot.

    Techniques Used: Infection, Mutagenesis, Transfection, Incubation, Imaging, Produced, Staining, Binding Assay, Western Blot

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects
    Article Snippet: .. siRNA transfections, quantitative PCR (qPCR) and dual luciferase assays The experiment shown in B was performed by transfection of stable siRNA or miRNA full sensor1 cell lines with 5 nM siRNA using lipofectamine 2000 (Invitrogen) in a six-well format using 450 000 cells per well. .. After 48 h both total RNA was collected using Trizol reagent (Invitrogen) and cell lysate for Dual luciferase assay, as described below.

    Modification:

    Article Title: Functionalized Asymmetric Bola-Type Amphiphiles for Efficient Gene and Drug Delivery
    Article Snippet: .. The Dulbecco’s modified Eagle’s medium (DMEM), opti-MEM culture medium, fetal bovine serum (FBS) and Lipofectamine® 2000 were purchased from Invitrogen Corp (Carlsbad, CA, USA). .. The 1 H NMR and 13 C NMR spectra were measured on a Bruker AM400 NMR spectrometer.

    Transfection:

    Article Title: A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects
    Article Snippet: .. siRNA transfections, quantitative PCR (qPCR) and dual luciferase assays The experiment shown in B was performed by transfection of stable siRNA or miRNA full sensor1 cell lines with 5 nM siRNA using lipofectamine 2000 (Invitrogen) in a six-well format using 450 000 cells per well. .. After 48 h both total RNA was collected using Trizol reagent (Invitrogen) and cell lysate for Dual luciferase assay, as described below.

    Article Title: Knockdown of Dinoflagellate Cellulose Synthase CesA1 Resulted in Malformed Intracellular Cellulosic Thecal Plates and Severely Impeded Cyst-to-Swarmer Transition
    Article Snippet: .. For each transfection sample, oligomer-Lipofectamine 2000 complexes were prepared by mixing 300 μl L medium, 300 μl 12 nmole antisense or scrambled ODN and 30 μl Lipofectamine Reagent 2000 (Invitrogen). .. The mixture was incubated for 30 min at room temperature (with rotation), before incubating with 300 μl spheroplasts (original culture: 800 ml, final cell number: ∼1.8 × 106 ) for another 1 h at room temperature (with gentle rotation).

    Article Title: PML isoform II plays a critical role in nuclear lipid droplet formation
    Article Snippet: .. Transfection Cells were transfected with cDNA and siRNA using Lipofectamine 2000 and Lipofectamine RNAiMAX (Invitrogen), respectively. .. They were analyzed for 2 (cDNA) and 3 d (siRNA) after transfection.

    Article Title: Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells
    Article Snippet: .. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for transfection following the manufacturer’s protocols. .. Human hepatoma cell lines Huh-7 and HepG2 cell were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM (containing 10% fetal bovine serum (FBS, Gibco, Invitrogen, Carlsbad, CA, USA) and 0.1% Penicilin-Streptomycin (Fischer Bioreagents, Pittsburgh, PA, USA)) at 37 °C in a 5% CO2 incubator.

    Luciferase:

    Article Title: A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects
    Article Snippet: .. siRNA transfections, quantitative PCR (qPCR) and dual luciferase assays The experiment shown in B was performed by transfection of stable siRNA or miRNA full sensor1 cell lines with 5 nM siRNA using lipofectamine 2000 (Invitrogen) in a six-well format using 450 000 cells per well. .. After 48 h both total RNA was collected using Trizol reagent (Invitrogen) and cell lysate for Dual luciferase assay, as described below.

    Plasmid Preparation:

    Article Title: A Method to Culture GABAergic Interneurons Derived from the Medial Ganglionic Eminence
    Article Snippet: .. We tried different combinations of DNA (pEGFP-N1 plasmid) and Lipofectamine-2000: either 0.1 or 0.2 μg of DNA were combined with either 0.35 or 0.5 μl of Lipofectamine-2000. ..

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  • 99
    Thermo Fisher lipofectamine 2000 transfection reagent
    Gene knockdown in vitro and in vivo cytotoxicity of fusogenic porous Si nanoparticle constructs. a siRNA knockdown results (via qRT-PCR) from Raw 264.7 macrophage cells incubated with nanoparticles for 24 h. Error bars indicate SD ( n = 6). The fusogenic formulations show substantial knockdown, comparable to standard <t>lipofectamine</t> <t>transfection</t> agent. No significant difference in knockdown efficiency is observed between the two fusogenic formulations (F-siIRF5-mPEG, F-siIRF5-CRV) and lipofectamine. *Significant difference (one-way ANOVA with Tukey’s HSD post hoc test, p -level
    Lipofectamine 2000 Transfection Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8704 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher transfection protocol
    Cellular function of the tagged TERT used for structural analysis. a , Western blot detection of ZZ-SS-TERT in TERT knock-out (KO) cells rescued by untagged TERT or ZZ-SS-TERT expression. Whole-cell extracts were probed using Strep antibody. HCT116 is the parental cell line. Lysate prepared from HEK 293T cells transiently transfected with ZZ-SS-TERT and hTR was used as a positive control (Ctrl). Tubulin was detected as a loading control. This experiment was performed only once to confirm the success of ZZ-SS-TERT incorporation into the HCT116 TERT KO cells. b , Telomeric restriction fragment analysis of HCT116 parental cells, TERT KO cells (before senescence), and TERT KO cells rescued with untagged or ZZ-SS-TERT transgene. Transgene-expressing cells were sampled at 31, 62 and 98 days <t>post-transfection</t> with transgene vectors. This experiment was performed twice with similar results. c , TRAP assay detection of telomerase activity in HCT116 parental cells, TERT KO cells, and TERT KO cells rescued with untagged TERT or ZZ-SS-TERT transgene. Whole-cell extracts were normalized by total protein concentration and assayed at 100, 30 or 10 ng of total protein per reaction. IC is an internal control. This experiment was repeated four times with similar results. d , Quantification of Q-TRAP assay detection of telomerase activity in HCT parental cells, TERT KO cells and TERT KO cells rescued with untagged TERT or ZZ-SS-TERT transgene. Error bars were calculated by taking the standard deviation of the average ΔCt from four different time points. Data points were shown as overlays. e , Direct primer-extension assay of telomerase after template-complementary oligonucleotide purification from extracts of TERT KO cells rescued by untagged TERT or ZZ-SS-TERT transgene. Assays were performed on clarified cell lysate (crude), flow-through (O-FT) and elution (OE) using equivalent amounts of cell extract. This experiment was performed only once to re-confirm the results in c .
    Transfection Protocol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gene knockdown in vitro and in vivo cytotoxicity of fusogenic porous Si nanoparticle constructs. a siRNA knockdown results (via qRT-PCR) from Raw 264.7 macrophage cells incubated with nanoparticles for 24 h. Error bars indicate SD ( n = 6). The fusogenic formulations show substantial knockdown, comparable to standard lipofectamine transfection agent. No significant difference in knockdown efficiency is observed between the two fusogenic formulations (F-siIRF5-mPEG, F-siIRF5-CRV) and lipofectamine. *Significant difference (one-way ANOVA with Tukey’s HSD post hoc test, p -level

    Journal: Nature Communications

    Article Title: Immunogene therapy with fusogenic nanoparticles modulates macrophage response to Staphylococcus aureus

    doi: 10.1038/s41467-018-04390-7

    Figure Lengend Snippet: Gene knockdown in vitro and in vivo cytotoxicity of fusogenic porous Si nanoparticle constructs. a siRNA knockdown results (via qRT-PCR) from Raw 264.7 macrophage cells incubated with nanoparticles for 24 h. Error bars indicate SD ( n = 6). The fusogenic formulations show substantial knockdown, comparable to standard lipofectamine transfection agent. No significant difference in knockdown efficiency is observed between the two fusogenic formulations (F-siIRF5-mPEG, F-siIRF5-CRV) and lipofectamine. *Significant difference (one-way ANOVA with Tukey’s HSD post hoc test, p -level

    Article Snippet: Fluorescent dyes Calcein (Sigma-Aldrich) and hydrophobic DiI (Life Technologies) were used and Lipofectamine® 2000 Transfection Reagent was obtained from Thermo Fisher Scientific.

    Techniques: In Vitro, In Vivo, Construct, Quantitative RT-PCR, Incubation, Transfection

    MiR-1180 is upregulated in WT, and the inhibition of miR-1180 suppressed WT cell proliferation. Notes: ( A ) The expression of miR-1180 in WT tissues compared with their adjacent non-cancerous tissues (n=30). ( B ) Transfection efficiency measured by the luciferase assay in the indicated cells. ( C ) qRT-PCR analysis of miR-1180 expression in the indicated cells. ( D ) The effect of miR-1180 inhibitor on the viability of WT as measured by the MTT assay. ( E ) Representative crystal violet-stained cell colonies formed by SK-NEP-1 cell lines, 8 days after inoculation. ( F ) The quantification of colonies of the SK-NEP-1 cells tested with colony formation. Data are presented as mean ± SD from three independent experiments with triple replicates per experiment. * p

    Journal: OncoTargets and therapy

    Article Title: MiR-1180-5p regulates apoptosis of Wilms’ tumor by targeting p73

    doi: 10.2147/OTT.S148684

    Figure Lengend Snippet: MiR-1180 is upregulated in WT, and the inhibition of miR-1180 suppressed WT cell proliferation. Notes: ( A ) The expression of miR-1180 in WT tissues compared with their adjacent non-cancerous tissues (n=30). ( B ) Transfection efficiency measured by the luciferase assay in the indicated cells. ( C ) qRT-PCR analysis of miR-1180 expression in the indicated cells. ( D ) The effect of miR-1180 inhibitor on the viability of WT as measured by the MTT assay. ( E ) Representative crystal violet-stained cell colonies formed by SK-NEP-1 cell lines, 8 days after inoculation. ( F ) The quantification of colonies of the SK-NEP-1 cells tested with colony formation. Data are presented as mean ± SD from three independent experiments with triple replicates per experiment. * p

    Article Snippet: Cell transfection was performed using Lipofectamine RNAiMax (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

    Techniques: Inhibition, Expressing, Transfection, Luciferase, Quantitative RT-PCR, MTT Assay, Staining

    Validation of the direct interaction between miRNA-1180 and p73 . Notes: ( A ) Bioinformatic analysis of the predicted interaction between miRNA-1180 and p73 . ( B ) The levels of luciferase activity following transfection of SK-NEP-1 cells with p73 3′-UTR-MT and p73 3′-UTR-WT sequences together with hsa-miRNA-1180 (* p

    Journal: OncoTargets and therapy

    Article Title: MiR-1180-5p regulates apoptosis of Wilms’ tumor by targeting p73

    doi: 10.2147/OTT.S148684

    Figure Lengend Snippet: Validation of the direct interaction between miRNA-1180 and p73 . Notes: ( A ) Bioinformatic analysis of the predicted interaction between miRNA-1180 and p73 . ( B ) The levels of luciferase activity following transfection of SK-NEP-1 cells with p73 3′-UTR-MT and p73 3′-UTR-WT sequences together with hsa-miRNA-1180 (* p

    Article Snippet: Cell transfection was performed using Lipofectamine RNAiMax (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

    Techniques: Luciferase, Activity Assay, Transfection

    Cellular function of the tagged TERT used for structural analysis. a , Western blot detection of ZZ-SS-TERT in TERT knock-out (KO) cells rescued by untagged TERT or ZZ-SS-TERT expression. Whole-cell extracts were probed using Strep antibody. HCT116 is the parental cell line. Lysate prepared from HEK 293T cells transiently transfected with ZZ-SS-TERT and hTR was used as a positive control (Ctrl). Tubulin was detected as a loading control. This experiment was performed only once to confirm the success of ZZ-SS-TERT incorporation into the HCT116 TERT KO cells. b , Telomeric restriction fragment analysis of HCT116 parental cells, TERT KO cells (before senescence), and TERT KO cells rescued with untagged or ZZ-SS-TERT transgene. Transgene-expressing cells were sampled at 31, 62 and 98 days post-transfection with transgene vectors. This experiment was performed twice with similar results. c , TRAP assay detection of telomerase activity in HCT116 parental cells, TERT KO cells, and TERT KO cells rescued with untagged TERT or ZZ-SS-TERT transgene. Whole-cell extracts were normalized by total protein concentration and assayed at 100, 30 or 10 ng of total protein per reaction. IC is an internal control. This experiment was repeated four times with similar results. d , Quantification of Q-TRAP assay detection of telomerase activity in HCT parental cells, TERT KO cells and TERT KO cells rescued with untagged TERT or ZZ-SS-TERT transgene. Error bars were calculated by taking the standard deviation of the average ΔCt from four different time points. Data points were shown as overlays. e , Direct primer-extension assay of telomerase after template-complementary oligonucleotide purification from extracts of TERT KO cells rescued by untagged TERT or ZZ-SS-TERT transgene. Assays were performed on clarified cell lysate (crude), flow-through (O-FT) and elution (OE) using equivalent amounts of cell extract. This experiment was performed only once to re-confirm the results in c .

    Journal: Nature

    Article Title: Cryo-EM structure of substrate-bound human telomerase holoenzyme

    doi: 10.1038/s41586-018-0062-x

    Figure Lengend Snippet: Cellular function of the tagged TERT used for structural analysis. a , Western blot detection of ZZ-SS-TERT in TERT knock-out (KO) cells rescued by untagged TERT or ZZ-SS-TERT expression. Whole-cell extracts were probed using Strep antibody. HCT116 is the parental cell line. Lysate prepared from HEK 293T cells transiently transfected with ZZ-SS-TERT and hTR was used as a positive control (Ctrl). Tubulin was detected as a loading control. This experiment was performed only once to confirm the success of ZZ-SS-TERT incorporation into the HCT116 TERT KO cells. b , Telomeric restriction fragment analysis of HCT116 parental cells, TERT KO cells (before senescence), and TERT KO cells rescued with untagged or ZZ-SS-TERT transgene. Transgene-expressing cells were sampled at 31, 62 and 98 days post-transfection with transgene vectors. This experiment was performed twice with similar results. c , TRAP assay detection of telomerase activity in HCT116 parental cells, TERT KO cells, and TERT KO cells rescued with untagged TERT or ZZ-SS-TERT transgene. Whole-cell extracts were normalized by total protein concentration and assayed at 100, 30 or 10 ng of total protein per reaction. IC is an internal control. This experiment was repeated four times with similar results. d , Quantification of Q-TRAP assay detection of telomerase activity in HCT parental cells, TERT KO cells and TERT KO cells rescued with untagged TERT or ZZ-SS-TERT transgene. Error bars were calculated by taking the standard deviation of the average ΔCt from four different time points. Data points were shown as overlays. e , Direct primer-extension assay of telomerase after template-complementary oligonucleotide purification from extracts of TERT KO cells rescued by untagged TERT or ZZ-SS-TERT transgene. Assays were performed on clarified cell lysate (crude), flow-through (O-FT) and elution (OE) using equivalent amounts of cell extract. This experiment was performed only once to re-confirm the results in c .

    Article Snippet: Genome engineering in HCT116 cells was performed using the manufacturer’s transfection protocol (Lipofectamine 3000, Thermo) with media exchange at 24 hours into selection with 200 μg/ml of G418 and 100 μg/ml of hygromycin.

    Techniques: Cell Function Assay, Western Blot, Knock-Out, Expressing, Transfection, Positive Control, TRAP Assay, Activity Assay, Protein Concentration, Standard Deviation, Primer Extension Assay, Purification, Flow Cytometry

    miR-145 and miR-497 inhibit EMT in NSCLC cells. a , b Western blot analysis for the expression of E-cadherin and vimentin in A549 cells treated with miR-145/miR-497 mimic or inhibitor. c , d The expression level of vimentin in H1299 cells transfected with miR-145/miR-497 mimic or inhibitor. (** P

    Journal: Cancer Cell International

    Article Title: miR-145 and miR-497 suppress TGF-β-induced epithelial–mesenchymal transition of non-small cell lung cancer by targeting MTDH

    doi: 10.1186/s12935-018-0601-4

    Figure Lengend Snippet: miR-145 and miR-497 inhibit EMT in NSCLC cells. a , b Western blot analysis for the expression of E-cadherin and vimentin in A549 cells treated with miR-145/miR-497 mimic or inhibitor. c , d The expression level of vimentin in H1299 cells transfected with miR-145/miR-497 mimic or inhibitor. (** P

    Article Snippet: The cells were transfected into H1299 and A549 cells by Lipofectamine 3000 (Thermo) according to the manufacturers’ protocol.

    Techniques: Western Blot, Expressing, Transfection

    Wound-healing assay in NSCLC. a , b Relative migrating areas were evaluated in A549 cells transfected with miR-145/miR-497 mimic or inhibitor. c , d After transfection of miR-145/miR-497 mimic or inhibitor, cells were treated with/without TGF-β and the relative migrating areas of H1299 cell were detected.(** P

    Journal: Cancer Cell International

    Article Title: miR-145 and miR-497 suppress TGF-β-induced epithelial–mesenchymal transition of non-small cell lung cancer by targeting MTDH

    doi: 10.1186/s12935-018-0601-4

    Figure Lengend Snippet: Wound-healing assay in NSCLC. a , b Relative migrating areas were evaluated in A549 cells transfected with miR-145/miR-497 mimic or inhibitor. c , d After transfection of miR-145/miR-497 mimic or inhibitor, cells were treated with/without TGF-β and the relative migrating areas of H1299 cell were detected.(** P

    Article Snippet: The cells were transfected into H1299 and A549 cells by Lipofectamine 3000 (Thermo) according to the manufacturers’ protocol.

    Techniques: Wound Healing Assay, Transfection

    miR-145 and miR-497 directly target MTDH. a , b The potential binding sites of miR-145/miR-497 and MTDH were predicted by bioinformatics tools Starbase v2.0 and Targetscan. c , d Relative luciferase activity in cells co-transfected with wild-type/mutant plasmid and miR-145/miR-497 mimic were evaluated. e – h The expression levels of MTDH in A549 and H1299 cells transfected with miR-145/miR-497 mimic or inhibitor. i , j The mRNA levels of MTDH in A549 and H1299 cells transfected with miR-145/miR-497 mimic or inhibitor. (** P

    Journal: Cancer Cell International

    Article Title: miR-145 and miR-497 suppress TGF-β-induced epithelial–mesenchymal transition of non-small cell lung cancer by targeting MTDH

    doi: 10.1186/s12935-018-0601-4

    Figure Lengend Snippet: miR-145 and miR-497 directly target MTDH. a , b The potential binding sites of miR-145/miR-497 and MTDH were predicted by bioinformatics tools Starbase v2.0 and Targetscan. c , d Relative luciferase activity in cells co-transfected with wild-type/mutant plasmid and miR-145/miR-497 mimic were evaluated. e – h The expression levels of MTDH in A549 and H1299 cells transfected with miR-145/miR-497 mimic or inhibitor. i , j The mRNA levels of MTDH in A549 and H1299 cells transfected with miR-145/miR-497 mimic or inhibitor. (** P

    Article Snippet: The cells were transfected into H1299 and A549 cells by Lipofectamine 3000 (Thermo) according to the manufacturers’ protocol.

    Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Expressing